Methods and products for manipulating uncoupling protein expression

ABSTRACT

The invention is based in part on the discovery that uncoupling proteins (UCPs) are expressed in the plasma membrane of rapidly dividing cells but not of growth arrested, chemotherapy resistant cells. It has also been found according to the invention that UCP is expressed in the lysosomal membrane under certain metabolic conditions. Thus the invention is methods, products, screening assays and kits relating to the manipulation of UCP expression within cellular and intracellular membranes.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/409,446, entitled “METHODS AND PRODUCTS FOR MANIPULATING UNCOUPLINGPROTEIN EXPRESSION” filed on Apr. 21, 2006, which is herein incorporatedby reference in its entirety. Application Ser. No. 11/409,446 is acontinuation of U.S. application Ser. No. 09/599,760, entitled “METHODSAND PRODUCTS FOR MANIPULATING UNCOUPLING PROTEIN EXPRESSION” filed onJun. 22, 2000, which is herein incorporated by reference in itsentirety. application Ser. No. 09/599,760 claims priority under 35U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/140,574,entitled “METHODS AND PRODUCTS FOR MANIPULATING UNCOUPLING PROTEINEXPRESSION IN THE PLASMA MEMBRANE” filed on Jun. 23, 1998, which isherein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the discovery that the cellularlocation of uncoupling protein (UCP) is altered in some cell types underdiffering metabolic states. In particular, the invention relates tomethods and products for manipulating UCP expression in variousmembranes. The invention also relates to methods of sensitizingresistant tumor cells to cytotoxic treatments by inducing expression ofUCP in the plasma membrane. The invention also relates to kits,compositions and screening assays.

BACKGROUND OF THE INVENTION

Normal tissue develops and is maintained by processes of cell divisionand cell death. In many diseases, such as cancer, diabetes mellitus TypeI, and autoimmune disease, the normal balance between cell division andcell death is disrupted causing either a rapid growth of unwanted andpotentially dangerous cells or a loss of cells which are essential tomaintaining the functions of tissue.

Cell division occurs by a process known as mitosis. During mitosisdividing cells use glucose cytolytically at an increased rate as theprimary source for energy (ATP) production in a process referred to asglycolysis (Brand, K A., and U. Hermfisse. 1997. Aerobic glycolysis byproliferating cells: a protective strategy against reactive oxygenspecies. Faseb J 11, no. 5:388-95). Glycolysis occurs in the cytosol andis required for mitochondrial energy production. An increased rate ofglycolysis occurs when cells divide, providing more of the ATP fromcytosolic glycolysis. Mitochondrial synthesis of ATP proceeds throughcoupling of electron transport-dependent oxido-reductive reactions toATP synthetase (oxidative phosphorylation) (Harper, M. E. 1997. Obesityresearch continues to spring leaks. Clinical Investigations in Medicine20, no. 4:239-244). During this process, a proton gradient is generatedby the pumping of protons out of the mitochondria (Himms-Hagen, J. 1992.Brown Adipose Tissue. Obesity, eds. P. Bjorntorp and B. N. Brodoff 1vols. J.B. Lippincott, Philadelphia. 1 pp), increasing mitochondrialmembrane potential. Uncoupling proteins (UCPs) reversibly uncoupleoxidative phosphorylation from electron transport in the mitochondriaand thereby can decrease mitochondrial membrane potential (Harper, M. E.1997. Obesity research continues to spring leaks. ClinicalInvestigations in Medicine 20, no. 4:239-244). Elevating glucoseconcentrations can increase mitochondrial membrane potential (Harper, M.E. 1997. Obesity research continues to spring leaks. ClinicalInvestigations in Medicine 20, no. 4:239-244). UCP and methods ofregulating or modulating UCP have been described in many publications,including for example U.S. Pat. Nos. 5,849,514; 5,849,581; 5,846,779;and 5,453,270.

Cell death is a physiologic process that ensures homeostasis ismaintained between cell production and cell turnover in self-renewingtissues and is essential to the proper functioning of the immune system.Physiological cell death occurs through the processes of apoptosis andnecrosis. The boundaries between these processes, once thought to bedistinct, have blurred with the explosion of information on the role ofcell death in development, tissue modeling, regenerative processes, andin the immune system. However, it is widely accepted that necrotic celldeath (sometimes called oncosis) typically results in the osmoticrupture of a cell, followed by an inflammatory response, while apoptoticdeath involves cell shrinkage, fragmentation of the cell, andphagocytosis of the fragments often without inflammation. Most cells diein a form of suicide characteristically apoptotic and tightly regulatedby complex signals (Zakeri, Z, W. Bursch, M. Tenniswood, and R. A.Lockshin. 1995. Cell Death: Programmed, apoptosis, necrosis, or other.Cell Death and Differentiation 2:87-96). Apoptotic cell death isparticularly important in the reticulo-endothelial system where thebalance between mitosis and cell death may determine the effectivenessand the nature of an immune response (Zakeri, Z., W Bursch, M.Tenniswood, and R. A. Lockshin. 1995. Cell Death: Programmed, apoptosis,necrosis, or other. Cell Death and Differentiation 2:87-96). Failureresults in autoimmune disease or in a lack of immune surveillance.

Inappropriate cell division or cell death results in seriouslife-threatening diseases. Diseases associated with increased celldivision include cancer and atherosclerosis. Disease resulting fromincreased cell death include AIDS, neurodegenerative diseases (e.g.,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,retinitis pigmentosa), aplastic anemia, atherosclerosis (e.g.,myocardial infarction, stroke, reperfusion injury), and toxin inducedliver disease. Many methods for treating these disorders have beenproposed Although these diseases share the common physiological trait ofeither excess cell division or premature cell death, strategies foridentifying potential therapeutic treatments have been individualizedrather than searching for a common mechanism. It would be desirable toidentify a common mechanism by which cell division could be interruptedor cell death could be promoted to treat all of these diseases.

SUMMARY OF THE INVENTION

The invention involves in some aspects the finding that uncouplingprotein (UCP) is present in membranes other than the mitochondria. Forinstance, it has been discovered that UCP is present in the plasmamembrane of rapidly dividing cells. It was found according to theinvention that the UCP in the plasma membrane plays an important role inthe signal which determines whether a cell will undergo cellulardivision, cellular differentiation or cellular death. This finding hasimportant implications for treating diseases associated with excessivecellular division, aberrant differentiation, and premature cellulardeath, e.g., for the treatment of cancers, autoimmune disease,degenerative diseases, regeneration etc. It has also been discoveredthat UCP is present in lysosomal membranes.

Several cell surface proteins have previously been identified as celldeath proteins. These proteins are believed to be involved in initiatinga signal which instructs the cell to die. Cell death proteins includefor example Fas/CD95 (Trauth et al., Science 245:301, 1989), tumornecrosis factor receptors, immune cell receptors such as CD40, OX40,CD27 and 4-1BB (Smith et al., Cell 76:959, 1994), and RIP (U.S. Pat. No.5,674,734). These proteins are believed to be important mediators ofcell death. These mediators, however, do not always instruct a cell todie. In some cases these mediators actually instruct a cell to undergocell division. The intracellular environment and particularly the statusof the proton motor force and source of fuel for mitochondrialmetabolism determines whether stimulation of the cell death protein willlead to a signal for death or cell division (co-pending U.S. patentapplication Ser. No. 09/277,575 incorporated herein by reference).

The invention involves the finding that another cell surface protein,UCP, also can regulate cell division by manipulating the manner in whichthe cell processes and utilizes energy. It was discovered according tothe invention that UCP is present on the plasma membrane of rapidlydividing cells but is not on the plasma membrane of growth arrested andchemotherapy resistant tumor cells. These findings have importantimplications on the ability to regulate cell division as well assensitivity and resistance to chemotherapeutic agents.

In one aspect the invention is a method for inhibiting plasma membraneUCP expression in a rapidly dividing cell. The method includes the stepof contacting a rapidly dividing cell with a plasma membrane targetedUCP inhibitor to inhibit plasma membrane UCP expression. In oneembodiment the method is a method for preventing cell division in arapidly dividing cell and wherein the loss of UCP activity in the plasmamembrane of the rapidly dividing cell prevents cell division.

The invention in another aspect is a method for inhibiting plasmamembrane UCP expression in a cell by contacting a cell with a plasmamembrane UCP inhibitor to inhibit plasma membrane UCP expression. In oneembodiment the plasma membrane UCP inhibitor is selected from the groupconsisting of a UCP binding peptide or molecule, an anti-UCP antibody, ahydrophobic nucleotide analog, UCP-inhibitor conjugated to a membraneattachment domain, and a non-omega-3 fatty acid.

The method may be used to induce cell death of a tumor cell. Thus in oneembodiment the rapidly dividing cell is a tumor cell. The tumor cell maybe in a subject, in which case the plasma membrane targeted UCPinhibitor is administered in vivo or ex vivo. The subject may also beadministered a cytotoxic anti-tumor therapy.

The method may also be performed on other rapidly dividing cells suchas, but not limited to a lymphocyte, a pancreatic β cell, or a bacteria.

In some embodiments the plasma membrane targeted UCP inhibitor is a UCPbinding peptide or molecule. In other embodiments the plasma membranetargeted UCP inhibitor is an anti-UCP antibody. In yet other embodimentsit is selected from the group consisting of a UCP antisense and dominantnegative UCP. In yet another embodiment the plasma membrane targeted UCPinhibitor is a nucleotide analog. When the rapidly dividing cell is atumor cell and the plasma membrane targeted UCP inhibitor is anucleotide analog the nucleotide analog may be, in some embodiments,targeted to specifically to the plasma membrane UCP. This can beaccomplished by linking the nucleotide analog to a cell surfacetargeting molecule or by manipulating the nucleotide analog or the cellto prevent uptake of the nucleotide analog by the cell. When the rapidlydividing cell is a lymphocyte, a pancreatic β cell, or a bacteria andthe plasma membrane targeted UCP inhibitor is a nucleotide analog thenucleotide analog the nucleotide analog may be delivered directly to thecell or may be targeted specifically to the plasma membrane UCP. In oneembodiment it is targeted specifically to the plasma membrane UCP.

In another aspect the invention is a composition of an UCP associatedwith a plasma or lysosomal membrane targeting molecule. The compositionmay optionally include a colloidal dispersion system, wherein the UCPand the plasma or lysosomal membrane targeting molecule are incorporatedinto the colloidal dispersion system. In one embodiment the colloidaldispersion system is a liposome.

In another aspect of the invention a composition of an UCP inhibitorassociated with a plasma or lysosomal membrane targeting molecule isprovided. The composition may optionally include a colloidal dispersionsystem, wherein the UCP inhibitor and the plasma or lysosomal membranetargeting molecule are incorporated into the colloidal dispersionsystem. In one embodiment the colloidal dispersion system is a liposome.

In some embodiments of the composition the plasma or lysosomal membranetargeted UCP inhibitor is a UCP binding peptide or molecule. In otherembodiments the plasma or lysosomal membrane targeted UCP inhibitor isan anti-UCP antibody or antibody fragment. In yet other embodiments itis selected from the group consisting of a UCP antisense and dominantnegative UCP. Optionally the UCP inhibitor is an anti-UCP antibodyconjugated to an anti-cell surface molecule antibody. In yet anotherembodiment the plasma or lysosomal membrane targeted UCP inhibitor is anucleotide analog.

A method for inducing cellular division in a growth arrested cell isprovided according to another aspect of the invention. The methodincludes the step of inducing expression of UCP in a plasma membrane ofa growth arrested cell under conditions in which the presence of the UCPwithin the plasma membrane of the growth arrested cell causes celldivision of the growth arrested cell. In another aspect, the inventionis a method for sensitizing a resistant tumor cell to a cytotoxictherapy. The method may be to performed by inducing expression of UCP inthe plasma membrane of a resistant tumor cell. The presence of the UCPwithin the plasma membrane of the resistant tumor cell renders theresistant tumor cell sensitive to cytotoxic therapy.

In one embodiment the method involves the step of contacting the growtharrested cell or resistant tumor cell with an UCP associated with aplasma membrane targeting molecule. In another embodiment the growtharrested cell or resistant tumor cell is a melanoma cell.

The invention also includes screening assays and kits relating to theabove-described methods and compositions. One screening assay of theinvention is a method for screening a tumor cell of a subject forsusceptibility to treatment with a chemotherapeutic agent. The methodinvolves the steps of isolating a tumor cell from a subject; anddetecting the presence of a UCP molecule in the plasma membrane of thetumor cell, wherein the presence of the UCP molecule in the plasmamembrane indicates that the tumor cell is susceptible to treatment witha chemotherapeutic agent.

In one embodiment the method comprises the step of contacting the tumorcell with a detection reagent that selectively binds to the plasmamembrane UCP molecule to detect the presence of the plasma membrane UCPmolecule. The plasma membrane UCP molecule may be a plasma membrane UCPmRNA. In that case the detection reagent is a nucleic acid thatselectively hybridizes to the plasma membrane UCP mRNA and wherein thecell is contacted with the detection reagent under conditions thatpermit selective hybridization of the nucleic acid to the plasmamembrane UCP mRNA. The plasma membrane UCP molecule may also be a plasmamembrane UCP polypeptide. In that case the detection reagent is a plasmamembrane UCP binding peptide and wherein the plasma membrane UCPpolypeptide is contacted with the detection reagent under conditionsthat permit selective binding of the plasma membrane UCP binding peptideto the plasma membrane UCP polypeptide. In one embodiment the plasmamembrane UCP binding peptide is an anti-plasma membrane UCP polypeptideantibody. In another embodiment the presence of the plasma membrane UCPpolypeptide is detected by contacting the tumor cell with a plasmamembrane UCP binding peptide attached to a solid support.

According to another aspect of the invention a method for screening asubject for the presence of rapidly dividing cells is provided. Themethod includes the steps of isolating a sample of cells from a subject;and, detecting the presence of a plasma membrane UCP molecule in theplasma membrane of the cell, wherein the presence of the plasma membraneUCP molecule is indicative of a rapidly dividing cell.

A kit for screening a tumor cell of a subject for susceptibility totreatment with a chemotherapeutic agent is provided according to anotheraspect of the invention. The kit includes a container housing a UCPmolecule detection reagent; and instructions for using the UCP moleculedetection reagent for detecting the presence of a UCP molecule on theplasma membrane of the tumor cell, wherein the presence of the plasmamembrane UCP molecule indicates that the cell is susceptible totreatment with a chemotherapeutic agent.

In one embodiment the kit also includes a container housing achemotherapeutic agent. In other embodiments the kit includes a panel ofchemotherapeutic agents, housed in separate compartments. In yet anotherembodiment the kit includes a UCP molecule detection reagent attached toa solid surface.

The invention in other aspects relates to a composition of a plasmamembrane targeted UCP inhibitor. In one embodiment the UCP inhibitor isa nucleotide or nucleotide analog. In another embodiment the nucleotideanalog is a purine analog. Preferably the purine analog is selected fromthe group consisting of guanosine diphosphate, 8-oxo-Adenosine,8-oxo-Guanosine, 8-fluoro-Adenosine, 8-fluoro-Guanosine,8-methoxy-Adenosine, 8-methoxy-Guanosine, 8-aza-Adenosine and8-aza-Guanosine, azacitidine, Fludarabine phosphate, 6-MP, 6-TG,azathiprine, allopurinol, acyclovir, gancylovir, deoxycoformycin, andarabinosyladienine (ara-A), guanosine diphosphate fucose, guanosinediphosphate-2-fluorofucose, guanosine diphosphate-.beta.L-2-aminofucose,guanosine diphosphate-D-arabinose and 2-aminoadenosine.

In another embodiment the nucleotide analog is a pyrimidine analog.Preferably the pyrimidine analog is selected from the group consistingof uracil, thymine, cytosine, 5-fluorouracil, 5-chlorouracil,5-bromouracil, dihydrouracil, 5-methylcytosine, 5-propynylthymine,5-propynyluracil and 5-propynylcytosine, 5-fluorocytosine, Floxuridine,uridine, thymine, 3′-azido-3′-deoxythymidine, 2-fluorodeoxycytidine,3-fluoro-3′-deoxythymidine; 3′-dideoxycytidin-2′-ene; and3′-deoxy-3′-deoxythymidin-2′-ene, and cytosine arabinoside.

In yet other embodiments the composition includes a pharmaceuticallyacceptable carrier. The composition may also include a colloidaldispersion system, wherein the plasma membrane UCP inhibitor isincorporated into the colloidal dispersion system. Optionally thecolloidal dispersion system is a liposome.

According to some embodiments the plasma membrane UCP inhibitor includesa hydrophobic moiety and optionally the plasma membrane UCP inhibitor isa modified nucleotide analog conjugated to a hydrophobic moiety. Inother embodiments the plasma membrane UCP inhibitor includes a membraneattachment domain, which is optionally conjugated to a nucleotide ornucleotide analog. In preferred embodiments the membrane attachmentdomain is a Type I membrane attachment domain, Type II membraneattachment domain, or Type III membrane attachment domain. In otherembodiments the membrane attachment domain is selected from the groupconsisting of P-Cadherin (FILPILGAVLALLLLLTLLALLLLV); CD2(IYLIIGICGGGSLLMVFVALLVFYIT); CD40 (ALVVIPIIFGILFAILLVLVFI); Contactin(ISGATAGVPTLLLGLVLPAP); IL-4 receptor (LLLGVSVSCIVILAVCLLCYVSIT);Mannose receptor (VAGVVIIVILLILTGAGLAAYFFY); M-CSF receptor(FLFTPVVVACMSIMALLLLLLLLLL); PDGFR .beta. chain(VVVISAILALVVLTIISLIILIMLWQKKPR); PDGFR .alpha. chain(ELTVAAAVLVLLVIVSISLIVLVVTW); P-Selectin (LTYFGGAVASTIGLIMGGTLLALL); RatThy-1 (VKCGGISLLVQNTSWLLLLLLSLSFLQATDFISL); TNFR-1(TVLLPLVIFFGLCLLSLLFIGLM); and VCAM-1 (LLVLYFASSLIIPAIGMIIYFAR).

The invention includes in other aspects a method for preventing ortreating a cancer by administering to a subject having a cancer theplasma membrane UCP inhibitor of the invention in effective amount totreat the cancer. Optionally an anti-tumor therapy may also beadministered.

A method for regulating lysosomal pH, is provided according to anotheraspect of the invention. The method includes the step of modifyinglysosomal UCP activity in a cell to regulate lysosomal pH. In someembodiments the cell is a T cell, a macrophage, or a neutrophil. Thelysosomal UCP activity may be modified by contacting the cell with alysosomal UCP inhibitor. In a preferred embodiment the lysosomal UCPinhibitor is selected from the group consisting of a dominant negativelysosomal UCP, and a lysosomal targeted binding peptide or molecule. Thelysosomal UCP activity also may be modified by contacting the cell witha lysosomal UCP activator.

The invention in other aspects relates to a method for treating orpreventing an infectious disease or a cancer by administering to asubject having or at risk of developing an infectious disease or acancer a lysosomal UCP inhibitor in an effective amount for treating orpreventing the infectious disease or cancer. In one embodiment thelysosomal UCP inhibitor is selected from the group consisting of adominant negative lysosomal UCP, and a lysosomal targeted bindingpeptide or molecule. The method optionally includes the step ofadministering an antigen to the subject. In one embodiment the antigenis selected from the group consisting of a viral, a bacterial, aparasitic, and a fungal antigen. In another embodiment the subject isinfected with an intracellular pathogen. Preferably the intracellularpathogen is an intracellular bacteria or an intracellular parasite.

According to another aspect of the invention a method for treatingautoimmune disease is provided. The method involves administering to asubject having autoimmune disease a lysosomal UCP activator in aneffective amount to prevent antigen presentation. In one embodiment thelysosomal UCP activator is a functional UCP or UCP fragment with alysosomal membrane targeting molecule.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of the human uncoupling (UCP-1)cDNA with GenBank Acc. no. U28480.

SEQ ID NO:2 is the predicted amino acid sequence of the translationproduct of to human uncoupling cDNA (UCP-1).

SEQ ID NO:3 is the nucleotide sequence of the human uncoupling (UCP-2)cDNA with GenBank Acc. no. U82819.

SEQ ID NO:4 is the predicted amino acid sequence of the translationproduct of human uncoupling cDNA (UCP-2).

SEQ ID NO:5 is the nucleotide sequence of the human uncoupling (UCP-3S)cDNA with GenBank Acc. no. U82818.

SEQ ID NO:6 is the predicted amino acid sequence of the translationproduct of human uncoupling cDNA (UCP-3S).

DETAILED DESCRIPTION

The invention relates in some aspects to the finding that UCP is presentin cellular membranes other than the mitochondrial membrane. Forinstance, UCP is expressed on the plasma membrane of rapidly dividingcells but not of growth arrested cells. This discovery has importantimplications for the regulation of cell death, division anddifferentiation.

In non-dividing cells of the body, mitochondria normally provide over90% of cellular ATP (Rolfe, D. F. S. & Brown, G. C. Cellular energyutilization and molecular origin of standard metabolic rate in mammals.Phys. Rev. 77, 731-758 (1997)). Mitochondrial ATP synthesis is fueled bythe oxidation of a variety of energy substrates (e.g., fatty acids,amino acids, and glucose), and it proceeds through the coupling of theelectron transport chain to the activity of ATP synthase. Coincident tothe transfer of electrons (or reducing equivalents) through componentsof the electron transport chain, protons are pumped into theintermembrane space, contributing to protonmotive force (Dp), across theinner membrane. Dp directly fuels mitochondrial ATP synthesis, as ATPsynthase is driven by proton movement from the intermembrane space tothe matrix. Uncoupling proteins (UCPs) present in the mitochondrialinner membrane have been proposed to cause mitochondrial proton leak andthereby reversibly uncouple oxidation from phosphorylation (Ricquier, D.& Bouillaud, F. The uncoupling protein homologues: UCP1, UCP2, UCP3,StUCP, and AtUCP. Biochem. J. 345, 161-179 (2000)).

Previously, it has been shown in co-pending U.S. patent application Ser.No. 09/277,575 (and in the Examples section below) that UCP is expressedin the mitochondria of growth arrested and chemotherapy resistant cellsbut not in rapidly dividing cells. In these studies, we comparedcharacteristics of mitochondrial metabolism in wild type anddrug-resistant cells, which die apoptotically and non-apoptotically,respectively (Bhushan, A. et al. Drug resistance results in alterationsin expression of immune recognition molecules and failure to express Fas(CD95). Immunol. Cell Biol. 76, 350-356 (1998)). Representative celllines stained with the mitochondrial dyes Mitotracker Green, whichquantitates mitochondria, and CmCX Ros Mitotracker Red (MolecularProbes, Eugene, Oreg.), which reflects mitochondrial membrane potential,revealed that the apoptotic resistant cells consistently have lowermitochondrial membrane potential as measured by the fluorescence uptakeof the Mitotracker Red dye. In order to quantitate and confirm theseresults, we conducted a full analysis of the overall metabolic kineticsof oxidative phosphorylation, using L1210 and L1210/DDP cells as modelsof apoptotic sufficient and apoptotic resistance, respectively. Leakdependent oxygen consumption for a given value of mitochondrial membranepotential (e.g., between 145 and 150 mV) was found to be markedly higherin the L1210/DDP cells than in the wild type cells. Thus leak isincreased in the resistant cell type. The overall kinetics of substrateoxidation and of phosphorylation were quite different between cell typesand were consistent with the results of the cell staining studiesdescribed above. Moreover, in flow cytometric experiments usingMitotracker Red dyes and single cell analysis, L1210, HL60, and U937cells consistently demonstrated a higher membrane potential than intheir respective drug resistant counterparts. In general these datademonstrate that mitochondrial uncoupling proteins may serve dualfunctions, i.e., to change carbon sources for fuel and to protect thecells from damage from reactive oxygen species.

It was then discovered according to the invention that UCP is alsoexpressed on other cellular membranes including the plasma membrane andthe lysosome. It was further discovered that the expression and activityof UCP in each of these distinct locations has an important impact onthe regulation of cellular growth and death. The metabolic shift frommitochondrial ATP production to cytosolic, glycolytic, and plasma ATPsynthesis likely occurs as a mechanism to protect newly synthesized andexposed DNA that could be damaged by products of mitochondrial oxygenconsumption. Thus to regulation of UCP expression and activity protectscells from DNA damaging agents, including radiation and/or chemotherapy.Additionally, lysosomal UCP expression is characteristic of rapidcellular growth.

These findings of the invention have important implications in thetreatment of disease and the study of cellular growth, death anddifferentiation, because it was not previously recognized that UCP wasexpressed in the plasma and lysosomal membranes and that plasma andlysosomal membrane UCP was involved in regulating various cellularfunctions.

Based on all these discoveries the invention includes in some aspectsmethods for increasing or decreasing the plasma membrane potential in amammalian cell. The ability to manipulate the plasma membrane potentialof a cell provides the ability to control the fate of the cell. When theplasma membrane potential of a cell is increased in a cell by increasingexpression of UCP in the plasma membrane, the cell is able to respond toa signal by rapid cell division or cell death depending on the signal.If the plasma membrane potential of a cell is decreased, however, byinhibiting plasma membrane UCP activity, the cell is growth arrested anddoes not respond to the same signals. When the lysosomal membranepotential of a cell, however, is increased by increasing expression ofUCP in the lysosomal membrane, the cell is growth arrested andrespiratory burst is inhibited. If the lysosomal membrane potential of acell is decreased by inhibiting lysosomal membrane UCP activity, thecell is able to respond to a signal by rapid cell division or cell deathdepending on the signal and respiratory burst is promoted. The inventionencompasses mechanisms for controlling these complex interactions toregulate the processes of cellular death and division and for respondingto microorganisms.

The methods of the invention have broad utility in regulating mammaliancell growth and death in vitro, in vivo and ex vivo. Because mammaliancells utilize the membrane potential and plasma and lysosomal membraneUCP in regulating their own growth and differentiation, any type ofmammalian cell can be manipulated according to the methods of theinvention.

The in vitro methods of the invention are useful for a variety ofpurposes. For instance, the methods of the invention may be useful foridentifying drugs which have an effect, such as a preventative effect,on cellular division or death by contacting cells which are caused bythe manipulations of the invention to undergo cellular division ordeath.

In addition to the in vitro methods, the methods of the invention may beperformed in vivo or ex vivo in a subject to manipulate one or more celltypes within the subject. An “ex vivo” method as used herein is a methodwhich involves isolation of a cell from a subject, manipulation of thecell outside of the body, and reimplantation of the manipulated cellinto the subject. The ex vivo procedure may be used on autologous orheterologous cells, but is preferably used on autologous cells. Inpreferred embodiments, the ex vivo method is performed on cells that areisolated from bodily fluids such as peripheral blood or bone marrow, butmay be isolated from any source of cells. When returned to the subject,the manipulated cell will be programmed for cell death or division,depending on the treatment to which it was exposed. Ex vivo manipulationof cells has been described in several references in the art, includingEngleman, E. G., 1997, Cytotechnology, 25:1; Van Schooten, W., et al.,1997, Molecular Medicine Today, June, 255; Steinman, R. M., 1996,Experimental Hematology, 24, 849; and Gluckman, J. C., 1997, Cytokines,Cellular and Molecular Therapy, 3:187. The ex vivo activation of cellsof the invention may be performed by routine ex vivo manipulation stepsknown in the art. In vivo methods are also well known in the art. Theinvention thus is useful for therapeutic purposes and also is useful forresearch purposes such as testing in animal or in vitro models ofmedical, physiological or metabolic pathways or conditions.

A subject as used herein means a human or non-human mammal, includingbut not limited to, a dog, cat, horse, cow, pig, sheep, goat, chicken,primate, rat, and mouse.

In one aspect the invention is a method for inhibiting plasma membraneUCP expression in a rapidly dividing cell. The method is accomplished bycontacting a rapidly dividing cell with a plasma membrane targeted UCPinhibitor to inhibit plasma membrane UCP expression. As discussed above,rapidly dividing cells express cell surface UCP but not (or low levelsof) mitochondrial or lysosomal UCP. Although the invention is notlimited to a particular mechanism, it is believed that rapidly dividingcells can process energy in the plasma membrane by converting energysources such as glucose to heat that is released. When the plasmamembrane UCP is inhibited according to the invention, the cell is unableto process the energy. Since there is no (or little) mitochondrial UCPpresent in these cells, the cells can no longer process the energysource and stop dividing, and eventually die.

A “rapidly dividing cell” as used herein is a cell which is undergoingmitotic growth. Such cells are well known in the art and include but arenot limited to tumor cells, lymphocytes (T/B cells), bacteria, andpancreatic β cells.

In some embodiments of the invention the rapidly dividing cell is atumor cell. The method is useful for inducing cell death in many typesof mammalian cells but is particularly useful for inducing cell death ina tumor cell. A “tumor cell” as used herein is a cell which isundergoing unwanted mitotic proliferation. A tumor cell when used in thein vitro aspects of the invention can be isolated from a tumor within asubject or may be part of an established cell line. A tumor cell in asubject may be part of any type of cancer. Cancers include but are notlimited to biliary tract cancer; brain cancer, including glioblastomasand medulloblastomas; breast cancer; cervical cancer; choriocarcinoma;colon cancer; endometrial cancer; esophageal cancer; gastric cancer;hematological neoplasms, including acute lymphocytic and myelogenousleukemia; multiple myeloma; AIDS associated leukemias and adult T-cellleukemia lymphoma; intraepithelial neoplasms, including Bowen's diseaseand Paget's disease; liver cancer; lung cancer; lymphomas, includingHodgkin's disease and lymphocytic lymphomas; neuroblastomas; oralcancer, including squamous cell carcinoma; ovarian cancer, includingthose arising from epithelial cells, stromal cells, germ cells andmesenchymal cells; pancreas cancer; prostate cancer; rectal cancer;sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma,fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi'ssarcoma, basocellular cancer and squamous cell cancer; testicularcancer, including germinal tumors (seminoma, non-seminoma[teratomas,choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer,including thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor.

As used herein the term “cell death” is used to refer to either of theprocesses of apoptosis or cell lysis. In both apoptosis and cell lysisthe cell dies but the processes occur through different mechanisms andwhen the cell is in a different metabolic state. Apoptosis is a processof cell death in which the cell undergoes shrinkage and fragmentation,followed by phagocytosis of the cell fragments. Apoptosis is well knownin the art and can be assessed by any art recognized method. For exampleapoptosis is easily determined using flow cytometry, which distinguishesbetween live and dead cells. Flow cytometry is described in more detailin the Examples below.

Diabetes mellitus, which encompasses both Type I (i.e., InsulinDependent Diabetes Mellitus (IDDM)) and Type II (i.e., Non-InsulinDependent Diabetes Mellitus (NIDDM)), is known to affect more than onehundred million individuals worldwide. Although the exact cause ofdiabetes is unclear it is believed that diabetes may arise from any of avariety of physiological conditions such as genetic syndromes, viralinfections, age related deterioration of structures responsible formaintaining the glycemic response, pancreatic disease, hormonalabnormalities, certain drugs or chemicals, insulin receptorabnormalities, etc. A “type I diabetic” is a subject who has diabetesmellitus caused by a destruction of beta cells in the pancreas. Type Idiabetics require daily insulin administration which may be reduced butnot altogether eliminated by careful restriction of diet.

Neither the genetic/environmental influences nor the inherent β cellcharacteristics that trigger immune-mediated destruction are completelyunderstood. However, two features that are pivotal in susceptibility toβ cell destruction are the expression of the cell surface molecule Fasand the metabolic state of the β cells. Fas can induce mitosis orapoptosis depending on the cell and the experimental circumstances.During the prediabetic stage of Type 1 diabetes, a β cell compensatoryhypersecretion of insulin occurs and this process is accompanied by cellsurface expression of the molecule Fas. When NOD mice, an animal modelfor Type 1 diabetes, are crossed with mice having the lpr mutation (Fasdeficient), the animals are resistant to disease. In addition,destruction of β cells in the NOD accelerates when Fas Ligand is placedon the insulin promotor.

It has been discovered according to the invention that changes in cellsurface expression of UCP contribute to β cell destruction or survival.β cell glucose-induced insulin secretion depends on increasedintracellular ATP. The mitochondrial synthesis of ATP occurs throughcoupling of electron transport-dependent oxido-reductive reactions toATP synthetase (oxidative phosphorylation). During this process, aproton gradient is generated by the pumping of protons out of themitochondria increasing mitochondrial membrane potential. UCPsreversibly uncouple oxidative phosphorylation from electron transportdecreasing mitochondrial membrane potential. Normal pancreatic β cellsare in an uncoupled state and do not express Fas or UCP on their cellsurface. As diabetes progresses to a first stage in which the patient issick but before the pancreatic β cell are destroyed, it is believed thatthe patients pancreatic β cells become coupled and express Fas and UCPon the cell surface. The disease then progresses to the stage whenpancreatic β cells begin to be killed. Before the cells are killed themetabolic state changes again to uncoupled and Fas is still expressed onthe surface. When the cell is in an uncoupled state and Fas is expressedon the cell surface the cell is killed as soon as Fas is engaged withoutthe need for any other agents.

A “type II diabetic” is a subject who has diabetes mellitus caused byabnormal insulin secretion and/or resistance to insulin action in targettissues. The physiological problem which occurs in a Type II diabetic isvery different than that which occurs in a type I diabetic. In type IIdiabetes the pancreatic β cells undergo excessive proliferation. It isdesirable to inhibit proliferation of these cells.

One method according to the invention for inducing pancreatic β celldeath in a Type II diabetic involves the step of contacting a pancreaticβ cell of a Type II diabetic with an amount of a plasma membranetargeted UCP inhibitor in an amount effective to induce pancreatic βcell death.

Autoimmune disease is a class of diseases in which an individuals ownantibodies react with host tissue or in which immune effector T cellsare autoreactive to endogenous self peptides and cause destruction oftissue. It is well established that MHC class II alleles act as majorgenetic elements in susceptibility to a variety of autoimmune diseases.These include rheumatoid arthritis, celiac disease, pemphigus vulgaris,and the prototype for autoimmune disease, systemic lupus erythematosus(SLE). The invention includes a new method for determining anindividuals susceptibility to developing autoimmune disease. As usedherein susceptibility to autoimmune disease indicates a likelihood of atleastgreater than the average of developing autoimmune disease, and insome embodiments 10% greater.

The methods of the invention also include methods for treating a subjecthaving autoimmune disease to reduce associated cell death. One method isbased on the ability to selectively remove γδ T cells which specificallyrecognize MHC class II HLA-DR on the surface of a self cell. When the γδT cells recognize a tissue having significant amounts of MHC class IIHLA-DR these T cells become activated and proliferate in order to killmore of the recognized cells. The methods of treatment are based on theconcept of eliminating the activated γδ T cells from the body. Thesecells can be removed by contacting a γδ T cell with an amount of aplasma membrane targeted UCP inhibitor in an amount effective to induceγδ T cell death. This selective killing of the γδ cells inhibits celldeath associated with autoimmune disease.

The invention is also useful for treating other diseases associated withrapidly dividing cells, such as rheumatoid arthritis and scleroderma.Rheumatoid arthritis is associated in its early stages with the rapiddivision of synoviocytes. This process is referred to a pannusformation. The rapidly dividing cells produce a substance that killsosteocytes leading to the hardening of the tissue. The plasma membranetargeted UCP inhibitors can be used to inhibit the proliferation of thesynoviocytes, thus preventing the hardening of the tissue. In a similarmanner plasma membrane targeted UCP inhibitors which are administered toskin of scleroderma subjects or subjects at risk of developingscleroderma can stop the rapid proliferation of skin cells which leadsto this disease.

The methods of the invention are also useful for inducing cell death ina rapidly dividing microorganism, such as a bacteria. Bacteria includeboth gram negative and gram positive bacteria. Such gram positivebacteria include, but are not limited to Pasteurella species,Staphylococci species, and Streptococcus species. Gram negative bacteriainclude, but are not limited to, Escherichia coli, Pseudomonas species,and Salmonella species. Specific examples of infectious bacteria includebut are not limited to: Helicobacter pyloris, Borelia burgdorferi,Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M.avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcusaureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeriamonocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira,Rickettsia, and Actinomyces israelli.

When plasma membrane UCP expression or function is inhibited inbacteria, the ability to rapidly divide is interrupted. It is believedthat bacterial cell death is accomplished in a similar manner to tumorcells when the plasma membrane UCP is inhibited. The methods of theinvention may also be combined with other anti-bacterial therapies andperformed on other types of rapidly dividing microorganisms.

The methods of inhibiting cell division in a rapidly dividing cell areaccomplished by contacting the cell with a plasma membrane targeted UCPinhibitor. A “plasma membrane targeted UCP inhibitor” as used herein isa molecule that inhibits the expression or activity of UCP in the plasmamembrane. Plasma membrane targeted UCP inhibitors include, for example,but are not limited to UCP binding peptides such as anti-UCP antibodies,UCP anti-sense nucleic acids, UCP dominant-negative nucleic acids andnucleotide analogs in an amount effect to inhibit plasma membrane UCPfunction.

Plasma membrane targeted UCP inhibitors include, for instance,nucleotides and nucleotide (purine and pyrimidine) analogs which havebeen modified to include a plasma membrane targeting sequence or aremembrane impermeable. Nucleotides and nucleotide analogs include but arenot limited to guanosine diphosphate (GDP). Purine analogs include butare not limited to guanosine diphosphate, 8-oxo-Adenosine,8-oxo-Guanosine, 8-fluoro-Adenosine, 8-fluoro-Guanosine,8-methoxy-Adenosine, 8-methoxy-Guanosine, 8-aza-Adenosine and8-aza-Guanosine, azacitidine, Fludarabine phosphate, 6-MP, 6-TG,azathiprine, allopurinol, acyclovir, gancylovir, deoxycoformycin, andarabinosyladienine (ara-A), guanosine diphosphate fucose, guanosinediphosphate-2-fluorofucose, guanosine diphosphate-.beta.L-2-aminofucose,guanosine diphosphate-D-arabinose and 2-aminoadenosine. Some examples ofpyrimidine analogues are uracil, thymine, cytosine, 5-fluorouracil,5-chlorouracil, 5-bromouracil, dihydrouracil, 5-methylcytosine,5-propynylthymine, 5-propynyluracil and 5-propynylcytosine,5-fluorocytosine, Floxuridine, uridine, thymine,3′-azido-3′-deoxythymidine, 2-fluorodeoxycytidine,3-fluoro-3′-deoxythymidine; 3′-dideoxycytidin-2′-ene; and3′-deoxy-3′-deoxythymidin-2′-ene, cytosine arabinoside. Other suchcompounds are known to those of skill in the art.

5-FU (e.g., “FLUOROURACIL” by Roche Labs., a division ofHoffman-LaRoche, Inc., Nutley, N.J. 07110) is a cytotoxicfluoropyrimidine antimetabolic commonly used in the palliativemanagement of carcinoma of the colon, rectum, breast, ovarian, cervix,bladder, stomach, liver and pancreas. 5-FU has been shown to havesynergistic interaction with other antineoplastic agents, interferons,and irradiation and is thus commonly used in combination therapy.5-Fluorouracil (5-FU) has been used continuously since its developmentin 1957 by Duusinski and Heidelberger (U.S. Pat. No. 2,802,005). 5-FUwas originally designed to work as an inhibitor of thymidylatesynthetase (TS). TS is the enzyme which converts deoxyuridine5′-O-monophosphate (dUMP) to deoxythymidine 5′-O-monophosphate (dTMP).It was previously believed that 5-FU retards tumor expansion by causingthymidine pools to become depleted in rapidly proliferating tumor cells.It has been found according to the invention that 5-FU that is targetedto the cell membrane and prevented from entering the cell can functionby inhibiting plasma membrane UCP.

Protocols for the administration of 5-FU for treatment of human cancerinvolve infusion of the drug for long periods of time. 5-FU that istaken up by the cell is rapidly metabolized and excreted with ahalf-life in vivo of about 18 minutes. The effectiveness of 5-FU ishampered by rapid metabolism and formation of 2-fluoro-.beta.-alanine(FBAL) which is neurotoxic and cardiotoxic. When 5-FU is used accordingto the invention it is either modified to prevent cell uptake ortargeted to the plasma membrane so that it is delivered to the plasmamembrane and is not taken up by the cell. Thus, use of 5-FU according tothe methods of the invention avoids the metabolic breakdown into toxiccompounds that causes the associated side effects.

Screening assays for determining the sensitivity of a cell to5-fluorouracil and its analogs have been described. Such an assay isdescribed in Anai H, Maehara Y, Kusumoto H, Kusumoto T, Sugimachi K,Oncology 1988; 45(3):144-7, “Sensitivity test for 5-fluorouracil and itsanalogues, 1-(2-tetrahydrofuryl)-5-fluorouracil,uracil/1-(2-tetrahydrofuryl)-5-fluorouracil (4:1) and1-hexylcarbamoyl-5-fluorouracil, using the subrenal capsule assay” Thispaper describes the testing of the chemosensitivity of human neoplastictissues using 5-fluorouracil (5-FU) and its analogues:1-(2-tetrahydrofuryl)-5-FU (FT), uracil/FT (UFT) and1-hexylcarbamoyl-5-FU (HCFU), and the in vivo subrenal capsule (SRC)assay. The to relative variation of tumor size (delta TS/TSo) wascalculated and the chemosensitivity was considered to be sensitive whendelta TS/TSo in the treated group was decreased to below −10%. Theresults of the study suggest that the SRC assay is useful for predictingthe effective drug among 5-FU and 5-FU analogues, for individualpatients with cancer. These assays can also be used with the plasmamembrane targeted or membrane impermeable forms of 5-FU to predict whichanalogs are useful.

Fludarabine phosphate (e.g., “FLUDARA” by Berlex Labs., Richmond, Calif.94804) is a purine analog antimetabolic commonly used in the treatmentof chronic lymphocytic leukemia (CLL). Floxuridine (e.g., “FUDR” byRoche Labs., a division of Hoffman-LaRoche, Inc., Nutley, N.J. 07110) isa cytotoxic drug commonly used in the palliative management ofgastrointestinal adenocarcinoma metastatic to the liver and is also usedfor treating brain, breast, head and neck cancers with liver metastases.The plasma membrane targeted or membrane impermeable forms of FLUDARAare also useful for the treatment of these cancers.

Thus nucleotides and nucleotide analogs can be modified to produceplasma membrane targeted UCP inhibitors by attaching a plasma membranetargeting sequence to the nucleotide or nucleotide analog. This can beaccomplished by linking the nucleotide analog to a cell surfacetargeting molecule. Several methods for linking molecules are describedbelow and others are known in the art. The nucleotide or nucleotideanalogs may also be modified such that it is membrane impermeable toprevent uptake of the nucleotide analog by the cell. By using compoundswhich are not taken up by a cell but simply act on the cell surface UCPmany of the toxic side effects associated with some of these drugs areavoided. The compounds will not have an effect on cells that do not haveUCP expressed in the plasma membrane, because they cannot access theintracellular UCP. Additionally, the compounds will not be metabolizedwithin cells to produce toxic compounds.

Plasma membrane targeted UCP inhibitors also include UCP bindingpeptides or molecules. The binding peptides or molecules can bedelivered directly to the cell to act on the plasma membrane UCP. Aslong as they are delivered by a mechanism which will not facilitateuptake of the molecule into the cell then the UCP binding peptide ormolecule will be targeted to the plasma membrane UCP as opposed to themitochondrial or lysosomal UCP. The UCP binding peptide or molecule mayalso be attached to a targeting molecule which targets the peptide ormolecule to the cell of interest, as discussed in more detail below.

The UCP binding peptides and molecules of the invention can beidentified using routine assays, such as the binding and activationassays described in the Examples and elsewhere throughout this patentapplication.

The UCP binding molecule is an isolated molecule. An isolated moleculeis a molecule that is substantially pure and is free of other substanceswith which it is ordinarily found in nature or in vivo systems to anextent practical and appropriate for its intended use. In particular,the molecular species are sufficiently pure and are sufficiently freefrom other biological constituents of host cells so as to be useful in,for example, producing pharmaceutical preparations or sequencing if themolecular species is a nucleic acid, peptide, or polysaccharide. Becausean isolated molecular species of the invention may be admixed with apharmaceutically-acceptable carrier in a pharmaceutical preparation, themolecular species may comprise only a small percentage by weight of thepreparation. The molecular species is nonetheless substantially pure inthat it has been substantially separated from the substances with whichit may be associated in living systems.

The UCP binding molecules may be isolated from natural sources orsynthesized or produced by recombinant means. Methods for preparing oridentifying molecules which bind to a particular target are well-knownin the art. Molecular imprinting, for instance, may be used for the denovo construction of macro molecular structures, such as peptides, whichbind to a particular molecule. See for example, Kenneth J. Shea,Molecular Imprinting of Synthetic Network Polymers: The De novoSynthesis of Molecular Binding In Catalytic Sites, Trip, to May 1994;Klaus, Mosbach, Molecular Imprinting, Trends in Biochem. Sci., 19(9),January 1994; and Wulff, G., In Polymeric Reagents and Catalysts (Ford,W. T., ed.) ACS Symposium Series No. 308, P. 186-230, Am. Chem. Soc.1986. Binding peptides, such as antibodies, may easily be prepared bygenerating antibodies to UCP (or obtained from commercial sources) or byscreening libraries to identify peptides or other compounds which bindto the UCP.

Many UCP antibodies are commercially available. These include but arenot limited to those antibodies commercially available from Santa CruzBiotechnology, Inc., e.g., UCP1 (m-17, sc-6529), UCP1 (C-17, sc-6528),UCP2 (A19, sc-6527), UCP2 (N19, sc-6526), UCP2 (c-20, sc-6525), and UCP3(C-20, sc-7756); antibodies commercially available from ResearchDiagnostics Inc e.g., Goat anti-UCP1 HUMAN/Mouse/Rat (cat#RDI-UCP1Cabg);Goat anti-UCP1 HUMAN/Mouse/Rat (cat#RDI-MUCP1Cabg); Goat anti-UCP2HUMAN/Mouse/Rat (cat#RDI-UCP2Nabg); Goat anti-UCP2 HUMAN/Mouse/Rat(cat#RDI-UCP2Cabg); Goat anti-UCP2 HUMAN/Mouse/Rat (cat#RDI-UCP2C1abg);Rabbit anti-Murine UCP1 (cat#RDI-MUCP12abrX); Rabbit anti-Murine UCP1(cat#RDI-MUCP19abrX); Rabbit anti-Murine UCP2 (cat#RDI-MUCP2abrX);Rabbit anti-Murine UCP2 (cat#RDI-MUCP2CabrX); Rabbit anti-human UCP2(cat#RDI-UCP2MabrX); UCP3L (see Boss, O et al (1997) FEBS Lett 408,38-42; Vidal-Plug A et al (1997) BBRC 235, 79-82); Rabbit anti-HUMANUCP3 (cat#RDI-UCP3abrX); Rabbit anti-HUMAN UCP3 (cat#RDI-UCP3CbrX);Rabbit anti-HUMAN UCP3 (cat#RDI-UCP3MabrX); Rabbit anti-Rat UCP3(cat#RDI-RTUCP3MabrX), etc.

Mimics of known binding molecules may also be prepared by known methods,such as (i) polymerization of functional monomers around a known bindingmolecule or the binding region of an antibody which also binds to thetarget (the template) that exhibits the desired activity; (ii) removalof the template molecule; and then (iii) polymerization of a secondclass of monomers in the void left by the template, to provide a newmolecule which exhibits one or more desired properties which are similarto that of the template. The method is useful for preparing peptides,and other binding molecules which have the same function as bindingpeptides, such as polysaccharides, nucleotides, nucleoproteins,lipoproteins, carbohydrates, glycoproteins, steroids, lipids and otherbiologically-active material can also be prepared. Thus a template, suchas a UCP binding antibody can be used to identify UCP inhibitors. It isnow routine to produce large numbers of inhibitors based on one or a fewpeptide sequences or sequence motifs. (See, e.g., Bromme, et al.,Biochem. J. 315:85-89 (1996); Palmer, et al., J. Med. Chem. 38:3193-3196(1995)). For example, if UCP is known to interact with protein X atposition Y, an inhibitor of UCP may be chosen or designed as apolypeptide or modified polypeptide having the same sequence as proteinX, or structural similarity to the sequence of protein X, in the regionadjacent to position Y. In fact, the region adjacent to the cleavagesite Y spanning residues removed by 10 residues or, more preferably 5residues, N-terminal and C-terminal of position Y, may be defined as a“preferred protein X site” for the choice or design of UCP inhibitors.Thus, a plurality of to UCP inhibitors chosen or designed to span thepreferred protein X binding site around position Y, may be produced,tested for inhibitory activity, and sequentially modified to optimize oralter activity, stability, and/or specificity.

The method is useful for designing a wide variety of biological mimicsthat are more stable than the natural counterpart, because they aretypically prepared by the free radical polymerization of functionalmonomers, resulting in a compound with a non-biodegradable backbone.Thus, the created molecules would have the same binding properties asthe UCP antibody but be more stable in vivo, thus preventing UCP frominteracting with components normally available in its nativeenvironment. Other methods for designing such molecules include, forexample, drug design based on structure activity relationships whichrequire the synthesis and evaluation of a number of compounds andmolecular modeling.

Binding molecules may also be identified by conventional screeningmethods, such as phage display procedures (e.g. methods described inHart et al., J. Biol. Chem. 269:12468 (1994)). Hart et al. report afilamentous phage display library for identifying novel peptide ligands.In general, phage display libraries using, e.g., M13 or fd phage, areprepared using conventional procedures such as those described in theforegoing reference. The libraries generally display inserts containingfrom 4 to 80 amino acid residues. The inserts optionally represent acompletely degenerate or biased array of peptides. Ligands having theappropriate binding properties are obtained by selecting those phagewhich express on their surface a ligand that binds to the targetmolecule. These phage are then subjected to several cycles ofreselection to identify the peptide ligand expressing phage that havethe most useful binding characteristics. Typically, phage that exhibitthe best binding characteristics (e.g., highest affinity) are furthercharacterized by nucleic acid analysis to identify the particular aminoacid sequences of the peptide expressed on the phage surface in theoptimum length of the express peptide to achieve optimum binding.

Alternatively, UCP binding molecules can be identified fromcombinatorial libraries. Many types of combinatorial libraries have beendescribed. For instance, U.S. Pat. No. 5,712,171 (which describesmethods for constructing arrays of synthetic molecular constructs byforming a plurality of molecular constructs having the scaffold backboneof the chemical molecule and modifying at least one location on themolecule to in a logically-ordered array); U.S. Pat. No. 5,962,412(which describes methods for making polymers having specificphysiochemical properties); and U.S. Pat. No. 5,962,736 (which describesspecific arrayed compounds).

To determine whether a molecule binds to the appropriate target anyknown binding assay may be employed. For example, in the case of apeptide that binds to the plasma membrane UCP the molecule may beimmobilized on a surface and then contacted with a labeled plasmamembrane UCP (or vice versa). The amount of plasma membrane UCP whichinteracts with the molecule or the amount which does not bind to themolecule may then be quantitated to determine whether the molecule bindsto plasma membrane UCP. A surface having a known molecule that binds toplasma membrane UCP such as a commercially available monoclonal antibodyimmobilized thereto may serve as a positive control. Several types ofcommercially available antibodies are described above.

Screening of molecules of the invention, also can be carried oututilizing a competition assay. If the molecule being tested competeswith the known monoclonal antibody, as shown by a decrease in binding ofthe known monoclonal antibody, then it is likely that the molecule andthe known monoclonal antibody bind to the same, or a closely related,epitope. Still another way to determine whether a molecule has thespecificity of the known monoclonal antibody is to pre-incubate theknown monoclonal antibody with the target with which it is normallyreactive, and then add the molecule being tested to determine if themolecule being tested is inhibited in its ability to bind the target. Ifthe molecule being tested is inhibited then, in all likelihood, it hasthe same, or a functionally equivalent, epitope and specificity as theknown monoclonal antibody.

By using the known UCP (and other target) monoclonal antibodies of theinvention, it is also possible to produce anti-idiotypic antibodieswhich can be used to screen other antibodies to identify whether theantibody has the same binding specificity as the known monoclonalantibody. Such anti-idiotypic antibodies can be produced usingwell-known hybridoma techniques (Kohler and Milstein, Nature, 256:495,1975). An anti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the known monoclonal antibodies. Thesedeterminants are located in the hypervariable region of the antibody. Itis this region which binds to a given epitope and, thus, is responsiblefor the specificity of the antibody. An anti-idiotypic antibody can beprepared by immunizing an animal with the known monoclonal antibodies.The immunized animal will recognize and respond to the idiotypicdeterminants of the immunizing known monoclonal antibodies and producean antibody to these idiotypic determinants. By using the anti-idiotypicantibodies of the immunized animal, which are specific for the knownmonoclonal antibodies of the invention, it is possible to identify otherclones with the same idiotype as the known monoclonal antibody used forimmunization. Idiotypic identity between monoclonal antibodies of twocell lines demonstrates that the two monoclonal antibodies are the samewith respect to their recognition of the same epitopic determinant.Thus, by using anti-idiotypic antibodies, it is possible to identifyother hybridomas expressing monoclonal antibodies having the sameepitopic specificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is theimage of the epitope bound by the first monoclonal antibody.

In one embodiment the binding peptides useful according to the inventionare antibodies or functionally active antibody fragments. Antibodies arewell known to those of ordinary skill in the science of immunology. Manyof the binding peptides described herein are available from commercialsources as intact functional antibodies, as described above. As usedherein, the term “antibody” means not only intact antibody molecules butalso fragments of antibody molecules retaining specific binding ability.Such fragments are also well known in the art and are regularly employedboth in vitro and in vivo. In particular, as used herein, the term“antibody” means not only intact immunoglobulin molecules but also thewell-known active fragments F(ab′)₂, and Fab. F(ab′)₂, and Fab fragmentswhich lack the Fc fragment of intact antibody, clear more rapidly fromthe circulation, and may have less non-specific tissue binding of anintact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

As is well-known in the art, the complementarity determining regions(CDRs) of an antibody are the portions of the antibody which are largelyresponsible for antibody specificity. The CDR's directly interact withthe epitope of the antigen (see, in general, Clark, 1986; Roitt, 1991).In both the heavy chain and the light chain variable regions of IgGimmunoglobulins, there are four framework regions (FR1 through FR4)separated respectively by three complementarity determining regions(CDR1 through CDR3). The framework regions (FRs) maintain the tertiarystructure of the paratope, which is the portion of the antibody which isinvolved in the interaction with the antigen. The CDRs, and inparticular the CDR3 regions, and more particularly the heavy chain CDR3contribute to antibody specificity. Because these CDR regions and inparticular the CDR3 region confer antigen specificity on the antibodythese regions may be incorporated into other antibodies or peptides toconfer the identical specificity onto that antibody or peptide.

According to one embodiment, the peptide of the invention is an intactsoluble monoclonal antibody in an isolated form or in a pharmaceuticalpreparation. An intact soluble monoclonal antibody, as is well known inthe art, is an assembly of polypeptide chains linked by disulfidebridges. Two principle polypeptide chains, referred to as the lightchain and heavy chain, make up all major structural classes (isotypes)of antibody. Both heavy chains and light chains are further divided intosubregions referred to as variable regions and constant regions. As usedherein the term “monoclonal antibody” refers to a homogenous populationof immunoglobulins which specifically bind to an epitope (i.e. antigenicdeterminant), e.g., of plasma membrane UCP, lysosomal UCP etc.

The peptide useful according to the methods of the present invention maybe an intact humanized a monoclonal antibody. A “humanized monoclonalantibody” as used herein is a human monoclonal antibody or functionallyactive fragment thereof having human constant regions and a binding CDR3region from a mammal of a species other than a human. Humanizedmonoclonal antibodies may be made by any method known in the art.Humanized monoclonal antibodies, for example, may be constructed byreplacing the non-CDR regions of a non-human mammalian antibody withsimilar regions of human antibodies while retaining the epitopicspecificity of the original antibody. For example, non-human CDRs andoptionally some of the framework regions may be covalently joined tohuman FR and/or Fc/pFc′ regions to produce a functional antibody. Thereare entities in the United States which will synthesize humanizedantibodies from specific murine antibody regions commercially, such asProtein Design Labs (Mountain View Calif.). For instance, a humanizedform of the Pharmingen anti-Fas antibody used in the attached Examplescould be easily prepared and used according to the methods of theinvention.

European Patent Application 0239400, the entire contents of which ishereby incorporated by reference, provides an exemplary teaching of theproduction and use of humanized monoclonal antibodies in which at leastthe CDR portion of a murine (or other non-human mammal) antibody isincluded in the humanized antibody. Briefly, the following methods areuseful for constructing a humanized CDR monoclonal antibody including atleast a portion of a mouse CDR. A first replicable expression vectorincluding a suitable promoter operably linked to a DNA sequence encodingat least a variable domain of an Ig heavy or light chain and thevariable domain comprising framework regions from a human antibody and aCDR region of a murine antibody is prepared. Optionally a secondreplicable expression vector is prepared which includes a suitablepromoter operably linked to a DNA sequence encoding at least thevariable domain of a complementary human Ig light or heavy chainrespectively. A cell line is then transformed with the vectors.Preferably the cell line is an immortalized mammalian cell line oflymphoid origin, such as a myeloma, hybridoma, trioma, or quadroma cellline, or is a normal lymphoid cell which has been immortalized bytransformation with a virus. The transformed cell line is then culturedunder conditions known to those of skill in the art to produce thehumanized antibody.

As set forth in European Patent Application 0239400 several techniquesare well known in the art for creating the particular antibody domainsto be inserted into the replicable vector. (Preferred vectors andrecombinant techniques are discussed in greater detail below.) Forexample, the DNA sequence encoding the domain may be prepared byoligonucleotide synthesis. Alternatively a synthetic gene lacking theCDR regions in which four framework regions are fused together withsuitable restriction sites at the junctions, such that double strandedsynthetic or restricted subcloned CDR cassettes with sticky ends couldbe ligated at the junctions of the framework regions. Another methodinvolves the preparation of the DNA sequence encoding the variable CDRcontaining domain by oligonucleotide site-directed mutagenesis. Each ofthese methods is well known in the art. Therefore, those skilled in theart may construct humanized antibodies containing a murine CDR regionwithout destroying the specificity of the antibody for its epitope.

Human monoclonal antibodies may be made by any of the methods known inthe art, such as those disclosed in U.S. Pat. No. 5,567,610, issued toBorrebaeck et al., U.S. Pat. No. 565,354, issued to Ostberg, U.S. Pat.No. 5,571,893, issued to Baker et al, Kozber, J. Immunol. 133: 3001(1984), Brodeur, et al., Monoclonal Antibody Production Techniques andApplications, p. 51-63 (Marcel Dekker, Inc, new York, 1987), and Boerneret al., J. Immunol., 147: 86-95 (1991). In addition to the conventionalmethods for preparing human monoclonal antibodies, such antibodies mayalso be prepared by immunizing transgenic animals that are capable ofproducing human antibodies (e.g., Jakobovits et al., PNAS USA, 90: 2551(1993), Jakobovits et al., Nature, 362: 255-258 (1993), Bruggermann etal., Year in Immuno., 7:33 (1993) and U.S. Pat. No. 5,569,825 issued toLonberg).

The binding peptides may also be functionally active antibody fragments.Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, 1. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions of theantibody, for example, are effectors of the complement cascade but arenot involved in antigen binding. An antibody from which the pFc′ regionhas been enzymatically cleaved, or which has been produced without thepFc′ region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. An isolated F(ab′)₂ fragment isreferred to as a bivalent monoclonal fragment because of its two antigenbinding sites. Similarly, an antibody from which the Fc region has beenenzymatically cleaved, or which has been produced without the Fc region,designated an Fab fragment, retains one of the antigen binding sites ofan intact antibody molecule. Proceeding further, Fab fragments consistof a covalently bound antibody light chain and a portion of the antibodyheavy chain denoted Fd (heavy chain variable region). The Fd fragmentsare the major determinant of antibody specificity (a single Fd fragmentmay be associated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

The terms Fab, Fc, pFc′, F(ab′)₂ and Fv are used consistently with theirstandard immunological meanings [Klein, Immunology (John Wiley, NewYork, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations ofModern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991)Essential Immunology, 7th Ed., (Blackwell to Scientific Publications,Oxford)].

In addition to the binding peptides and molecules, the invention alsoencompasses the use of antisense oligonucleotides that selectively bindto a plasma membrane UCP nucleic acid molecule, and dominant negativeUCP to reduce the expression of plasma membrane UCP. Antisenseoligonucleotides are useful, for example, for inhibiting plasma membraneUCP in a cell in which it is ordinarily expressed in the plasmamembrane.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide which hybridizes under physiologicalconditions to DNA comprising a particular gene or to an RNA transcriptof that gene and, thereby, inhibits the transcription of that geneand/or the translation of the mRNA. The antisense molecules are designedso as to hybridize with the target gene or target gene product andthereby, interfere with transcription or translation of the targetmammalian cell gene. Those skilled in the art will recognize that theexact length of the antisense oligonucleotide and its degree ofcomplementarity with its target will depend upon the specific targetselected, including the sequence of the target and the particular baseswhich comprise that sequence. The antisense must be a unique fragment. Aunique fragment is one that is a ‘signature’ for the larger nucleicacid. It, for example, is long enough to assure that its precisesequence is not found in molecules outside of the UCP gene. As will berecognized by those skilled in the art, the size of the unique fragmentwill depend upon its conservancy in the genetic code. Thus, some regionsof SEQ ID NO:1, 3, and 5, will require longer segments to be uniquewhile others will require only short segments, typically between 12 and32 base pairs (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 and 32 bases long).

It is preferred that the antisense oligonucleotide be constructed andarranged so as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon the known sequence of a gene that is targeted forinhibition by antisense hybridization, or upon allelic or homologousgenomic and/or cDNA sequences, one of skill in the art can easily chooseand synthesize any of a number of appropriate antisense molecules foruse in accordance with the present invention. In order to besufficiently selective and potent for inhibition, such antisenseoligonucleotides should comprise at least 7 and, more preferably, atleast 15 consecutive bases which are complementary to the target. Mostpreferably, the antisense oligonucleotides comprise a complementarysequence of 20-30 bases. Although oligonucleotides may be chosen whichare antisense to any region of the gene or RNA (e.g., mRNA) transcripts,in preferred embodiments the antisense oligonucleotides arecomplementary to 5′ sites, such as translation initiation, transcriptioninitiation or promoter sites, that are upstream of the gene that istargeted for inhibition by the antisense oligonucleotides. In addition,3′-untranslated regions may be targeted. Furthermore, 5′ or 3′ enhancersmay be targeted. Targeting to mRNA splice sites has also been used inthe art but may be less preferred if alternative mRNA splicing occurs.In at least some embodiments, the antisense is targeted, preferably, tosites in which mRNA secondary structure is not expected (see, e.g.,Sainio et al., Cell Mol. Neurobiol., (1994) 14(5):439-457) and at whichproteins are not expected to bind. The selective binding of theantisense oligonucleotide to a mammalian target cell nucleic acideffectively decreases or eliminates the transcription or translation ofthe mammalian target cell nucleic acid molecule. Reduction intranscription or translation of the nucleic acid molecule is desirablein preparing an animal model for further defining the role played by themammalian target cell nucleic acid in modulating an adverse medicalcondition.

The invention also includes the use of a “dominant negative plasmamembrane UCP” polypeptide. A dominant negative polypeptide is aninactive variant of a protein, which, by interacting with the cellularmachinery, displaces an active protein from its interaction with thecellular machinery or competes with the active protein, thereby reducingthe effect of the active protein. For example, a dominant negativereceptor which binds a ligand but does not transmit a signal in responseto binding of the ligand can reduce the biological effect of expressionof the ligand. Likewise, a dominant negative catalytically-inactivekinase which interacts normally with target proteins but does notphosphorylate the target proteins can reduce phosphorylation of thetarget proteins in response to a cellular signal. Similarly, a dominantnegative transcription factor which binds to a promoter site in thecontrol region of a gene but does not increase gene transcription canreduce the effect of a normal transcription factor by occupying topromoter binding sites without increasing transcription.

The end result of the expression of a dominant negative polypeptide asused herein in a cell is a reduction in plasma membrane expressed UCP.One of ordinary skill in the art can assess the potential for a dominantnegative variant of a protein, and using standard mutagenesis techniquesto create one or more dominant negative variant polypeptides. Forexample, one of ordinary skill in the art can modify the sequence of theplasma membrane UCP by site-specific mutagenesis, scanning mutagenesis,partial gene deletion or truncation, and the like. See, e.g., U.S. Pat.No. 5,580,723 and Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Theskilled artisan then can test the population of mutagenized polypeptidesfor diminution in a selected and/or for retention of such an activity,or simply for presence in the plasma membrane. Other similar methods forcreating and testing dominant negative variants of a protein will beapparent to one of ordinary skill in the art.

In addition to the plasma membrane UCP inhibitors of the invention anadditional method for inhibiting plasma membrane UCP involvestransfection of a cell with a UCP2 expression vector. Transfection ofcells with a UCP2 expression vector causes decreased expression andactivity of plasma membrane UCP. Thus it is useful to transfect arapidly dividing cell with a UCP2 expression vector in order to inducegrowth arrest of the cell.

Additionally a UCP nucleic acid can be delivered to a cell such that theUCP peptide will be expressed in the plasma membrane of the cell. TheUCP expression vectors and other relevant expression vectors describedherein can be prepared and inserted into cells using routine proceduresknown in the art. These procedures are set forth below in more detail.“UCP nucleic acid”, as used herein, refers to a nucleic acid moleculewhich: (1) hybridizes under stringent conditions to a nucleic acidhaving the sequence of SEQ ID NO:1, 3, and 5 and (2) codes for a UCPpolypeptide. The preferred UCP nucleic acid has the nucleic acidsequence of SEQ ID NO:1, 3, and 5 (the nucleic acids encoding the humanUCP-1, UCP-2, and UCP-3 polypeptides respectively). The UCP nucleicacids may be intact UCP nucleic acids which include the nucleic acidsequence of Sequence ID No.:1, 3, and 5 as well as homologs and allelesof a nucleic acid having the sequence of SEQ ID NO:1, 3, and 5. IntactUCP nucleic acids further embrace nucleic acid molecules which differfrom the sequence of SEQ ID NO:1, 3, and 5 in codon sequence due to thedegeneracy of the genetic code. The UCP nucleic acids of the inventionmay also be functionally equivalent variants, analogs and fragments ofthe foregoing nucleic acids. “Functionally equivalent”, in reference toa UCP nucleic acid variant, analog or fragment, refers to a nucleic acidthat codes for a UCP polypeptide that is capable of functioning as anUCP. The invention further embraces complements of the foregoing nucleicacids or of unique fragments of the foregoing nucleic acids. Suchcomplements can be used, for example, as antisense nucleic acids forinhibiting the expression of UCP in a cell in order to create anexperimental model of a cell in which UCP is not expressed.

The UCP nucleic acid molecules can be identified by conventionaltechniques, e.g., by identifying nucleic acid sequences which code forUCP polypeptides and which hybridize to a nucleic acid molecule havingthe sequence of SEQ ID NO:1, 3, and 5 under stringent conditions. Theterm “stringent conditions”, as used herein, refers to parameters withwhich the art is familiar. More specifically, stringent conditions, asused herein, refer to hybridization at 65° C. in hybridization buffer(3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serumalbumin, 2.5 mM NaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15Msodium chloride/0.15M sodium citrate, pH 7; SDS is sodium dodecylsulphate; and EDTA is ethylenediaminetetraacetic acid. Afterhybridization, the membrane to which the DNA is transferred is washed at2×SSC at room temperature and then at 0.1×SSC/0.1×SDS at 65° C.

There are other conditions, reagents, and so forth which can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions and, thus, they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of the UCP nucleic acid of the invention. Theskilled artisan also is familiar with the methodology for screeningcells and libraries for the expression of molecules, such as UCP, whichcan be isolated, followed by purification and sequencing of thepertinent nucleic acid molecule. In screening for UCP nucleic acidsequences, a Southern blot may be performed using the foregoingconditions, together with a radioactive probe. After washing themembrane to which the DNA is finally transferred, the membrane can beplaced against x-ray film to detect the radioactive signal.

In general, homologs and alleles typically will share at least 40%nucleotide identity with SEQ ID NO:1, 3, and 5; in some instances, willshare at least 50% nucleotide identity; and in still other instances,will share at least 60% nucleotide identity. The preferred homologs haveat least 70% sequence homology to SEQ ID NO:1, 3, and 5. More preferablythe preferred homologs have at least 80% and, most preferably, at least90% sequence homology to SEQ ID NO:1, 3, and 5.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the naturally occurring nucleicacid that codes for the human UCP polypeptide. For example, serineresidues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Eachof the six codons is equivalent for the purposes of encoding a serineresidue. Thus, it will be apparent to one of ordinary skill in the artthat any of the serine-encoding nucleotide codons may be employed todirect the protein synthesis apparatus, in vitro or in vivo, toincorporate a serine residue. Similarly, nucleotide sequence tripletswhich encode other amino acid residues include, but are not limited to,CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG(arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT(asparagine codons); and ATA, ATC and ATT (isoleucine codons). Otheramino acid residues may be encoded similarly by multiple nucleotidesequences. Thus, the invention embraces degenerate nucleic acids thatdiffer from the naturally occurring nucleic acids in codon sequence dueto the degeneracy of the genetic code.

The UCP nucleic acid, in one embodiment, is operably linked to a geneexpression sequence which directs the expression of the UCP nucleic acidwithin a eukaryotic cell. The “gene expression sequence” is anyregulatory nucleotide sequence, such as a promoter sequence orpromoter-enhancer combination, which facilitates the efficienttranscription and translation of the UCP nucleic acid to which it isoperably linked. The gene expression sequence may, for example, be amammalian or viral promoter, such as a constitutive or induciblepromoter. Constitutive mammalian promoters include, but are not limitedto, the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, and β-actin.Exemplary viral promoters which function constitutively in eukaryoticcells include, for example, promoters from the simian virus, papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcomavirus, cytomegalovirus, the long terminal repeats (LTR) of moloneyleukemia virus and other retroviruses, and the thymidine kinase promoterof herpes simplex virus. Other constitutive promoters are known to thoseof ordinary skill in the art. The promoters useful as gene expressionsequences of the invention also include inducible promoters. Induciblepromoters are expressed in the presence of an inducing agent. Forexample, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined UCP nucleic acid. The gene expression sequences optionallyinclude enhancer sequences or upstream activator sequences as desired.

Preferably, the UCP nucleic acid of the invention is linked to a geneexpression sequence which permits expression of the UCP nucleic acid inthe plasma membrane of a cell, e.g. a resistant tumor cell. A sequencewhich permits expression of the UCP nucleic acid in the plasma membraneof a tumor cell is one which is selectively active in the particulartumor cell and thereby causes the expression of the UCP nucleic acid inthese cells. Those of ordinary skill in the art will be able to easilyidentify promoters that are capable of expressing a UCP nucleic acid ina tumor cell based on the type of tumor cell, as well as other knowncells.

The UCP nucleic acid sequence and the gene expression sequence are saidto be “operably linked” when they are covalently linked in such a way asto place the transcription and/or translation of the UCP coding sequenceunder the influence or control of the gene expression sequence. If it isdesired that the UCP sequence be translated into a functional protein,two DNA sequences are said to be operably linked if induction of apromoter in the 5′ gene expression sequence results in the transcriptionof the UCP sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the UCP sequence, or (3) interfere with theability of the corresponding RNA transcript to be translated into aprotein. Thus, a gene expression sequence would be operably linked to aUCP nucleic acid sequence if the gene expression sequence were capableof effecting transcription of that UCP nucleic acid sequence such thatthe resulting transcript might be translated into the desired protein orpolypeptide.

The invention also encompasses methods for inducing cellular division ina growth arrested cell by expressing an UCP in a plasma membrane of agrowth arrested cell under conditions in which the presence of the UCPwithin the plasma membrane of the growth arrested cell causes celldivision of the growth arrested cell.

There are many ways to induce expression of UCP in a plasma membrane ofa cell. For instance, it is possible to insert an intact UCP, orfunctional fragment thereof, into a plasma membrane using deliveryvehicles such as liposomes. UCP is a naturally occurring plasma membraneprotein having several transmembrane spanning regions including manyhydrophobic residues. Proteins of this type can spontaneously insertinto a biological membrane in an aqueous environment. See, e.g., U.S.Pat. No. 5,739,273 (which is hereby incorporated by reference)describing properties of bacteriorhodopsin C helix, a transmembranespanning protein. The UCP can be inserted in to a biological membraneconsistent with the methods described in U.S. Pat. No. 5,739,273 forinserting bacteriorhodopsin C into a membrane, including in lipidvesicles and by modification of various residues to increase thehydrophobicity of the molecule, without altering the function.Additionally UCP can be conjugated to a molecule which will insert inthe membrane, causing the UCP to also insert in the membrane.

As set forth in U.S. Pat. No. 5,739,273 cell membranes are composedmainly of phospholipids and proteins, both containing hydrophobic andhydrophilic groups. The lipids orient themselves into an orderly bilayerconfiguration within the membrane core with the hydrophobic chainsfacing toward the center of the membrane while the hydrophilic portionsare oriented toward the outer and inner membrane surfaces. The proteinsare dispersed throughout the lipid layer, in some instances protrudingthrough the surface of the membrane or extending from one side of themembrane to the other with some of the hydrophobic residues being buriedin the interior of the lipid bilayer.

U.S. Pat. No. 5,739,273 teaches that a synthetic polypeptide maintainingthe to characteristics of a native polypeptide by including ahydrophobic alpha-helical transmembrane region containing one or moreacidic or basic amino acids can be generated. Preferably, the aminoacids are aspartic acid, glutamic acid, lysine, arginine or histidine.This is based on the teachings of Popot and Engelman, Biochem.29:4031-4037 (1990), that recently proposed a two-stage model of helixformation for transmembrane proteins in which the alpha-helices firstinsert into the lipid bilayer and then assemble into a tertiarystructure that includes interactions with other intramembranealpha-helices of the protein or with alpha-helices of other polypeptidesin the membrane.

The UCP insertion into the membrane can be enhanced using lipidvesicles. Lipid vesicles such as micelles can be formed by the additionof phospholipids to achieve a specific ratio of protein to phospholipid.The orientation of the chimeric protein components of the micelles canbe controlled also, so that the micelles have an outer surface which ispredominantly composed of the phospholipid moieties or predominantlycomposed of the protein moieties. The size of the micelles may also becontrolled by varying the detergent employed, the nature of the addedphospholipid, or the phospholipid/protein ratio.

Generally, the size of liposomes directly affects the rate at which theyare cleared from the bloodstream. For example, smaller liposomes andnegatively charged liposomes appear to be more stable and accumulate inthe spleen and liver. Thus, the micelles and liposomes can be tailoredto remain in the bloodstream for a desired period and to be delivered tospecific organs. For example, small micelles can be formed with an outersurface exhibiting a predominantly negative charge from thephosphoinositol moiety.

UCP proteins include the intact native UCP in an isolated form as wellas functionally active fragments and variants thereof. The native UCPprotein has an amino acid sequence as presented in SEQ ID NO:2.

The relationship between plasma membrane UCP and cell surface Fasexpression is important to the methods of the invention. When UCP isexpressed on the plasma membrane Fas is also expressed on the cellsurface in the plasma membrane. When UCP is not expressed on the plasmamembrane Fas generally is transported to intracellular stores. When UCPand Fas are on the surface, engagement of Fas sends a signal to the cellinstructing the cell to undergo cellular division. If a chemotherapeuticagent is added then the signal is changed to a signal which instructsthe cell to undergo apoptosis. When Fas and UCP are not expressed on thecell surface, the cell is growth arrested and the cell is resistant tochemotherapy if the cell is a tumor cell.

An “apoptotic chemotherapeutic agent” as used herein is a group ofmolecules which function by a variety of mechanisms to induce apoptosisin rapidly dividing cells. Apoptotic chemotherapeutic agents are a classof chemotherapeutic agents which are well known to those of skill in theart. Chemotherapeutic agents include those agents disclosed in Chapter52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and theintroduction thereto, 1202-1263, of Goodman and Gilman's “ThePharmacological Basis of Therapeutics”, Eighth Edition, 1990,McGraw-Hill, Inc (Health Professions Division), incorporated herein byreference. Suitable chemotherapeutic agents may have various mechanismsof action. The classes of suitable chemotherapeutic agents include (a)Alkylating Agents such as nitrogen mustard (e.g. mechlorethamine,cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines andmethylmelamines (e.g. hexamethylmelamine, thiotepa), alkyl sulfonates(e.g. busulfan), nitrosoureas (e.g. carmustine which is also known asBCNU, lomustine which is also known as CCNU semustine which is alsoknown as methyl-CCNU, chlorozoticin, streptozocin), and triazines (e.g.dicarbazine which is also known as DTIC); (b) Antimetabolites such asfolic acid analogs (e.g. methotrexate), pyrimidine analogs (e.g.5-fluorouracil floxuridine, cytarabine, and azauridine and its prodrugform azaribine), and purine analogs and related materials (e.g.6-mercaptopurine, 6-thioguanine, pentostatin); (c) Natural Products suchas the vinca alkaloids (e.g. vinblastine, Vincristine),epipodophylotoxins (e.g. etoposide, teniposide), antibiotics (e.g.dactinomycin which is also known as actinomycin-D, daunorubicin,doxorubicin, bleomycin, plicamycin, mitomycin, epirubicin, which is4-epidoxorubicin, idarubicin which is 4-dimethoxydaunorubicin, andmitoxanthrone), enzymes (e.g L-asparaginase), and biological responsemodifiers (e.g. Interferon alfa); (d) Miscellaneous Agents such as theplatinum coordination complexes (e.g. cisplatin, carboplatin),substituted ureas (e.g. hydroxyurea), methylhydiazine derivatives (e.g.procarbazine), adreocortical suppressants (e.g. mitotane,aminoglutethimide) taxol; (e) Hormones and Antagonists to such asadrenocorticosteroids (e.g. prednisone or the like), progestins (e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate), estrogens (e.g. diethyestilbestrol, ethinyl estradiol, and thelike), antiestrogens (e.g. tamoxifen), androgens (e.g. testosteronepropionate, fluoxymesterone, and the like), antiandrogens (e.g.flutamide), and gonadotropin-releasing hormone analogs (e.g. leuprolide)and (F) DNA damaging compounds such as adriamycin.

Additionally, the plasma membrane targeted UCP inhibitor can be used incombination with other therapies, such as radiation therapy. When acombination of therapies are used the effective amount to achieve thedesired result, inhibition of cell proliferation will be less. This maybe advantageous because it will reduce or eliminate any side effectsassociated with high concentrations of the individual therapies. Aparticularly preferred combination therapy is a plasma membrane targetedUCP inhibitor and radiation therapy. It is believed that radiation alsocontributes to the inhibition of plasma membrane UCP. Radiationsensitive cells are those cells that express plasma membrane UCP andradioresistant cells do not express plasma membrane UCP. The inventionalso includes methods of treating radioresistant cells by inducing UCPexpression in the plasma membrane of these cells as described below andtreating them with radiation.

In addition to the methods of manipulating cells, the invention is alsouseful for screening cells such as tumor cells to determine if thosecells are susceptible to cellular division or cellular death, alone orin conjunction with treatment with a chemotherapeutic agent or othercell signal and kits for performing these screening assays. Thescreening method can be accomplished by isolating a tumor cell from asubject and detecting the presence of a UCP molecule in the plasmamembrane of the tumor cell. The presence of a plasma membrane UCPindicates the tumor cell is susceptible to treatment with achemotherapeutic agent.

The screening methods are particularly useful for determining if a tumoris sensitive to a chemotherapeutic agent. A tumor, however, mayinitially be sensitive to a particular chemotherapeutic agent and thenas the therapy progresses the tumor may become resistant to thatchemotherapeutic agent. The methods of the invention can be used toprevent the tumor from becoming resistant to a chemotherapeutic agentduring therapy. The method involves the steps of administering to asubject in need of such to treatment a chemotherapeutic agent and aplasma membrane UCP activator in a combined amount effective to kill thetumor. The plasma membrane UCP activator causes the plasma membrane UCPto become or remain activated, preventing the cell from developing achemotherapy resistant phenotype. As the cell is held in this coupledstate Fas is expressed on the surface and the chemotherapeutic agent canstimulate Fas mediated apoptosis.

A plasma membrane UCP activator as used herein is compound that inducesthe uncoupling function of a UCP molecule that is already expressed inthe plasma membrane or causes a functional UCP to be expressed orinserted into the plasma membrane. These compounds include but are notlimited to glucose, structural analogs of glucose, inhibitors ofnucleotides and nucleotide analogs, and omega 3 fatty acids.

The combined amount of UCP activator and apoptotic chemotherapeuticagent effective to inhibit growth of the tumor cell is that amounteffective to induce apoptosis of the tumor cell when the plasma membranepotential is decreased (as a result of the activation of the UCP). Aneffective amount means that amount necessary to delay the onset of,inhibit the progression of, halt altogether the onset or progression ofthe particular condition being treated. In general, an effective amountfor treating a tumor cell is that amount necessary to halt theproliferation of the cell. In one embodiment, the effective amount isthat amount necessary to kill the cell. In general, an effective amountfor treating cancer will be that amount necessary to favorably affectmammalian cancer cell proliferation in-situ. An effective mount of theplasma membrane UCP activator is that amount sufficient to render thecell sensitive to chemotherapy. When administered to a subject,effective amounts will depend, of course, on the particular conditionbeing treated; the severity of the condition; individual patientparameters including age, physical condition, size and weight;concurrent treatment; frequency of treatment; and the mode ofadministration. These factors are well known to those of ordinary skillin the art and can be addressed with no more than routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment.

In some cases the screening assay may indicate that the tumor is mostlyresistant to a chemotherapeutic agent. Resistant tumors may also betreated by the methods of the invention. One aspect of the inventioninvolves the discovery that resistant tumors cells have a plasmametabolic state in which electron transport is coupled from oxidativephosphorylation in the plasma membrane because of the absence of UCP. Itwas discovered according to the invention that by altering the metabolicstate of the tumor cell and thereby causing electron transport to becoupled to oxidative phosphorylation in the plasma membrane it ispossible to cause the resistant cell to revert such that it becomessensitive to chemotherapy. The method is performed by expressing a UCPin the plasma membrane of the resistant tumor cell under conditions inwhich the presence of the UCP within the plasma membrane causes celldivision of the tumor cell.

Other screening assays can be performed according to the invention toidentify the presence of rapidly dividing cells in a subject. The assaycan be performed by isolating a sample of cells from a subject; and,detecting the presence of a plasma membrane UCP molecule in the plasmamembrane of the cell, wherein the presence of the plasma membrane UCPmolecule is indicative of a rapidly dividing chemotherapy sensitivecell.

Optionally, a targeting mechanism can be used to target the UCPinhibitor or activator to a specific cell type. It is desirable in manyinstances to specifically target a cell type to increase the efficiencyand specificity of administration of the plasma membrane UCP inhibitoror activator, thus avoiding the effects that damage or destroy unrelatedcells. Thus, an efficient delivery system which would enable thedelivery of such drugs specifically to target cells would increase theefficacy of treatment and reduce the associated “side effects” of suchtreatments.

Methods of targeting drugs and other compounds to target cells are wellknown in the art. One method of targeting involves antibody or receptortargeting. Receptor or antibody targeting involves linking the UCPinhibitor or activator to a ligand or an antibody which has an affinityfor a receptor or cell surface molecule expressed on the desired targetcell surface. Using this approach, the UCP inhibitor or activator isintended to adhere to the target cell following formation of aligand-receptor or antibody-cell surface antigen complex on the cellsurface. The type of receptor or antibody used to target the cell willdepend on the specific cell type being targeted.

A target molecule may be attached by a peptide or other type of bondsuch as a sulfhydryl or disulfide bond. Targeting molecules aredescribed, for instance in U.S. Pat. No. 5,849,718 as well as many otherreferences.

In general the targeting moiety is coupled to the UCP inhibitor oractivator. The molecules may be directly coupled to one another, such asby conjugation or may be indirectly coupled to one another where, forexample, the targeting moiety is on the surface of a liposome and theUCP inhibitor or activator is contained within the liposome. If themolecules are linked to one another, then the targeting moiety iscovalently or noncovalently bound to the UCP inhibitor or activator in amanner that preserves the targeting specificity of the targeting moiety.As used herein, “linked” or “linkage” means two entities are bound toone another by any physiochemical means. It is important that thelinkage be of such a nature that it does not impair substantially theeffectiveness of the UCP inhibitor or activator or the bindingspecificity of the targeting moiety. Keeping these parameters in mind,any linkage known to those of ordinary skill in the art may be employed,covalent or noncovalent. Such means and methods of linkage are wellknown to those of ordinary skill in the art.

Linkage according to the invention need not be direct linkage. Thecomponents of the compositions of the invention may be provided withfunctionalized groups to facilitate their linkage and/or linker groupsmay be interposed between the components of these compositions tofacilitate their linkage. In addition, the components of the presentinvention may be synthesized in a single process, whereby the componentscould be regarded as one in the same entity. For example, a targetingmoiety specific for a tumor cell could be synthesized together with theUCP inhibitor or activator. These and other modifications are intendedto be embraced by the present invention.

Specific examples of covalent bonds include those wherein bifunctionalcross-linker molecules are used. The cross-linker molecules may behomobifunctional or heterobifunctional, depending upon the nature of themolecules to be conjugated. Homobifunctional cross-linkers have twoidentical reactive groups. Heterobifunctional cross-linkers have twodifferent reactive groups that allow sequential conjugation reaction.Various types of commercially available cross-linkers are reactive withone or more of the following groups: primary amines, secondary amines,sulfhydriles, carboxyls, carbonyls and carbohydrates.

Non-covalent methods of conjugation also may be used to join thetargeting moiety and the UCP inhibitor or activator. Non-covalentconjugation may be accomplished by direct or indirect means includinghydrophobic interaction, ionic interaction, intercalation, binding tomajor or minor grooves of a nucleic acid and other affinityinteractions.

Covalent linkages may be noncleavable in physiological environments orcleavable in physiological environments, such as linkers containingdisulfide bonds. Such molecules may resist degradation and/or may besubject to different intracellular transport mechanisms. One of ordinaryskill in the art will be able to ascertain without undue experimentationthe preferred bond for linking the targeting moiety and the UCPinhibitor or activator, based on the chemical properties of themolecules being linked and the preferred characteristics of the bond.

For indirect linkage, the targeting moiety may be part of a particle,such as a liposome, which targets the liposome to tissues expressing thetumor cell. The liposome, in turn, may contain the UCP inhibitor oractivator. The manufacture of liposomes containing UCP inhibitor oractivator is fully described in the literature. Many are based uponcholesteric molecules as starting ingredients and/or phospholipids. Theymay be synthetically derived or isolated from natural membranecomponents. Virtually any hydrophobic substance can be used, includingcholesteric molecules, phospholipids and fatty acids preferably ofmedium chain length (12C-20C). Preferred are naturally occurring fattyacids of between 14 and 18 carbons in length. These molecules can beattached to the UCP inhibitor or activator of the invention, with thelipophilic anchor inserting into the membrane of a liposome and the UCPinhibitor or activator tethered on the surface of the liposome fortargeting the liposome to the cell.

In some embodiments the UCP activators and inhibitors are targeted tothe lysosome. Native UCP molecules include a lysosomal targetingsequence which likely plays a role in targeting the UCP to the lysosomalmembrane under the appropriate cellular conditions. This sequence is aNCT motif. This same motif can be used to target activators andinhibitors to the lysosomal membrane. Once the UCP is in the lysosome,it can function to regulate pH. Numerous publications describe targetingof nucleic acids and peptides to lysosomal compartments within cells.See, for instance, Pauly, D., et al., Gene Ther., 5 (4), 473-80 (1998);Gerlier, D., Cell Biol. Int., 18 (5), 315-20 (1994); Gough, N. et al.,J. Cell Sci., 112 (PT23) 4257-4269 (1999); Calvo, P., et al., J. Biol.Chem., 274 (18), 12780-9 (1999); Williams, M., and Fukuda, M., J. Cell.Biol., 111 (3), 955-66, (1990); Matthews, P., et al., J. Cell Biol., 118(5), 1027-40 (1992); Sandoval, I., et al., J. Biol. Chem., 269 (9),6622-31 (1994); Blagoveshchenskaya, A., et al., J. Biol. Chem., 273 (5),2729-37 (1998); and Peters, C., EMBO J., 9 (11), 3497-506, (1990), theentire contents of which are hereby incorporated by reference. One classof compounds that is known to accumulate in lysosomes is compoundshaving a pKa such that it is uncharged at physiological pH but becomesprotonated in the more highly acidic lysosomal compartment. Examples arechloroquine and N-basic substituted 3-hydroxypyridin-4-one orally activechelators. Other protein based reagents may also be known to traffick tolysosomes preferentially and are useful according to the invention.

Native UCP also contains a mitochondrial targeting domain. This motif isdescribed in Schleiff and McBride is a three unit motif.

Plasma membrane targeting sequences include hydrophobic moieties andmembrane attachment domains. Hydrophobic moieties are well known in theart. A “membrane attachment domain,” as used herein, refers to a domainthat spans the width of a cell/plasma membrane, or any part thereof, andthat functions to attach a UCP inhibitor to a cell membrane. Membraneattachment domains useful in the invention are those domains thatfunction to attach a UCP inhibitor to a plasma membrane of an eukaryoticcell or the outer membrane of a prokaryotic cell. One skilled in the artunderstands that an appropriate membrane attachment domain is selectedbased on the type of cell in which the membrane-bound fusion protein isto be expressed.

A variety of naturally occurring and synthetic membrane attachmentdomains derived from eukaryotic and prokaryotic cell surface proteinsare useful in the invention. For use in higher eukaryotic cells such asmammalian cells, a membrane attachment domain can be, for example, themembrane-spanning region of an integral membrane protein such as a cellsurface receptor or cell adhesion molecule. Membrane attachment domainsuseful in the invention can be derived, for example, from cell surfacereceptors including growth factor receptors such as platelet derivedgrowth factor receptor, epidermal growth factor receptor or fibroblastgrowth factor receptor; hormone receptors; cytokine receptors and T cellreceptor. Membrane attachment domains useful in the invention also canbe derived from cell adhesion molecules such as cadherins, integrins,selectins and members of the immunoglobulin superfamily; as well asother integral membrane proteins such as CD antigens. The amino acidsequences of exemplary membrane attachment domains are described herein(see, also Pigott and Power, The adhesion Molecule Facts Book San Diego:Academic Press, Inc. (1993) and Barclay et al., The Leukocyte AntigenFacts Book San Diego: Academic Press, Inc. (1993), each of which isincorporated herein by reference). If desired, the fusion protein caninclude the cytosolic domain, or portion thereof, of the heterologousprotein from which the membrane attachment domain is derived.

Type I membrane attachment domains are transmembrane sequences of about25 hydrophobic amino acid residues usually followed by a cluster ofbasic amino acids Amino acids that are usually excluded from suchmembrane attachment domains include Asn, Asp, Glu, Gln, His, Lys andArg, although where the domains form a multimeric complex in themembrane, there can be charged residues present. The orientation of atype I membrane attachment domain is such that the amino-terminalportion is extracellular. Such type I membrane attachment domains can bederived, for example, from CD2, CD40 or the IL-4 receptor.

Type II membrane attachment domains are transmembrane domains useful inthe invention. The orientation of a type II membrane attachment domainis such that the carboxy-terminal portion is extracellular. Examples oftype II membrane attachment domains include the transmembrane domain ofCD72.

A membrane attachment domain of the invention also can be aphosphatidylinositol-glycan (PI-G) anchor, which is attached to thecarboxy-terminal residue of a protein. A PI-G anchor can be derived, forexample, from human placental alkaline phosphatase (HPAP), and canfunction to anchor a fusion protein to the cell surface (see, forexample, Whitehorn et al., Biotechnology 13:1215-1219 (1995), which isincorporated herein by reference). PI-G-anchored molecules have a signalsequence at their carboxy-terminus that is cleaved off and replaced bythe PI-G anchor. The residues at the PI-G attachment site andimmediately following are typically small amino acids such as Ala, Asn,Asp, Gly, Cys or Ser. After the attachment residue, there is ahydrophobic sequence of about 10 to 20 residues starting 7-10 residuesafter the attachment point. Such hydrophobic PI-G-signal sequencesgenerally lack the basic charged residues found in type I membraneattachment domains.

Other domain include polymers or dendrimers that will not typicallypenetrate or traffick through a cell membrane. The nucleotides or otherUCP inhibitors or activators may be singly or multiply bound to thepolymer. Polymers that are charged or having high molecular weight canbe discovered or developed to have minimize cell permeability. Polymersor dendrimers having a net negative charge may be preferred. Polymerssuch as polyacrylates, polymethacrylates, heparin, dextran sulfate,hyaluronic acid and other charged polymers are also useful. Positivelycharged polymers such as polyvinylpyrrolidone, polylysine orpolyethyleneimine may also be useful. Neutral or uncharged polymers suchas polyethylene glycols, pluronics and tetronics may also be useful.Additionally, negatively charged moieties such as phosphates,thiophosphates, and other known oligonucleotide modifications may beuseful.

Type III membrane attachment domains, or segments thereof, also can beuseful in the invention. Such type III membrane attachment domains arederived from eukaryotic cell surface molecules that cross the lipidbilayer numerous times. A membrane attachment domain useful in theinvention can be, for example, one or more transmembrane domains derivedfrom MDR1, a G-protein linked receptor or a protein of the rhodopsinsuperfamily. Exemplary membrane attachment domains include but are notlimited to P-Cadherin (FILPILGAVLALLLLLTLLALLLLV); CD2(IYLIIGICGGGSLLMVFVALLVFYIT); CD40 (ALVVIPIIFGILFAILLVLVFI); Contactin(ISGATAGVPTLLLGLVLPAP); IL-4 receptor (LLLGVSVSCIVILAVCLLCYVSIT);Mannose receptor (VAGVVIIVILLILTGAGLAAYFFY); M-CSF receptor(FLFTPVVVACMSIMALLLLLLLLLL); PDGFR .beta. chain(VVVISAILALVVLTIISLIILIMLWQKKPR); PDGFR .alpha. chain(ELTVAAAVLVLLVIVSISLIVLVVTW); P-Selectin (LTYFGGAVASTIGLIMGGTLLALL); RatThy-1 (VKCGGISLLVQNTSWLLLLLLSLSFLQATDFISL); TNFR-1(TVLLPLVIFFGLCLLSLLFIGLM); and VCAM-1 (LLVLYFASSLIIPAIGMIIYFAR).

The term “heterologous,” as used herein in reference to a membraneattachment domain operatively fused to a UCP inhibitor, means a membraneattachment domain derived from a source other than the gene encoding theUCP inhibitor. A heterologous membrane attachment domain can besynthetic or can be encoded by a gene distinct from the gene encodingthe UCP inhibitor to which it is fused.

The term “operatively fused,” as used herein in reference to a UCPinhibitor and a heterologous membrane attachment domain, means that theUCP inhibitor and membrane attachment domain are fused in the correctreading frame such that, under appropriate conditions, a full-lengthfusion protein is expressed. One skilled in the art would recognize thatsuch a fusion protein can comprise, for example, an amino-terminal UCPinhibitor operatively fused to a carboxyl-terminal heterologous membraneattachment domain or can comprise an amino-terminal heterologousmembrane attachment domain operatively fused to a carboxyl-terminal UCPinhibitor.

The term “membrane-bound,” as used herein in reference to a fusionprotein means stably attached to a cellular membrane. The term “fusionprotein,” as used herein, means a hybrid protein including a syntheticor heterologous amino acid sequence.

The plasma membrane UCP inhibitor is delivered to the rapidly dividingcell in an amount effective to inhibit rapid mitotic growth. Aneffective amount to inhibit rapid mitotic growth is that amount which issufficient to slow down, or inhibit altogether the proliferation and/orrapid mitotic growth of the rapidly dividing cell.

As used herein, the term “dissipation of cellular proton motor force”refers to the relative amount of protons in the cell. It can be assessedby measuring plasma, lysosomal, or mitochondrial membrane potentialdepending on the UCP being studied. As used herein “plasma membranepotential” is the pressure on the inside of the plasma membrane measuredrelative to the extracellular fluid which is created by the generationand dissipation of charge within the cell. The “lysosomal membranepotential” is the pressure on the inside of the lysosomal membranemeasured relative to the cytoplasma which is created by the generationand dissipation of charge within the lysosome. The plasma or lysosomalmembrane potential is maintained by the energy generating system of theplasma or lysosomal membrane respectively. In most tissues electrontransport is coupled to oxidative phosphorylation resulting in theproduction of ATP from glucose. UCPs can cause the reversible uncouplingof electron transport and oxidative phosphorylation, which leads to adecrease in the mitochondrial membrane potential, or as discoveredherein the plasma or lysosomal membrane potential. Other tissue, oftenreferred to as the immuno-privileged tissue such as the brain, testis,ovary, eye, and pancreatic β cells, express UCPs which cause electrontransport to be uncoupled to oxidative phosphorylation under normalconditions. In these tissues glucose cannot be converted to ATP whilethe UCP is active because of the uncoupling and the energy produced isconverted into other energy forms such as heat and released. If themetabolic processing systems in these tissues are caused to undergocoupling the membrane potential would increase.

The absolute levels of the plasma membrane potential vary depending onthe cell or tissue type. As used herein an “increase in plasma orlysosomal membrane potential” is an increase relative to the normalstatus of the cell being examined and results from the prevention ofdissipation of proton motor force with respect to plasma or lysosomalrespectively. “Prevention” as used herein refers to a decrease orreduction in the amount of dissipation that would ordinarily occur inthe absence of the stimulus applied according to the methods of theinvention to cause coupling. If electron transport and oxidativephosphorylation are normally uncoupled within the plasma or lysosomalmembrane of the cell then the baseline potential will be relatively lowand when the ATP generating systems are coupled an increase in plasma orlysosomal membrane potential from that baseline level is observed.Likewise, a “decrease in plasma or lysosomal membrane potential” is adecrease relative to the normal status of the cell being examined andresults from the dissipation of proton motor force. If electrontransport and oxidative phosphorylation are normally coupled within thecell then the baseline potential will be relatively high and when theATP generating systems are uncoupled a decrease in plasma membranepotential from that baseline level is observed. Plasma or lysosomalmembrane ATP synthase is likely the source of ATP for the plasma orlysosomal membrane UCP.

Changes in plasma or lysosomal membrane potential can be assessed by anymethod known in the art for making such measurements. For example theplasma or lysosomal membrane potential may be assessed using the wellknown comet assay, where whole cells are electrophoresed on an agarosegel and examined for the presence of a tail. Alternatively it may bemeasured using electrodes placed on opposite sides of the membrane.Plasma or lysosomal membrane potential may also be measuredcytometrically by incubating cells for approximately 20 minutes at roomtemperature with a plasma or lysosomal membrane specific fluorescentprobe. The aggregation state and consequently the fluorescence emissionof fluorescent probe changes as the plasma or lysosomal membranepotential is altered. Flow cytometry permits the examination of morethan one, for instance eight, fluorescent markers concurrently. Thismethod is described in more detail in the Examples section below Inaddition to examining the plasma or lysosomal membrane potential,studies can be performed to determine the rate of glucose utilizationand oxidation and measurements of proton leak can be assessed by atop-down elasticity analysis, each of which is described in more detailin the Examples below. An additional method for determining if a cellexpresses active UCP in the plasma or lysosomal membrane is an adherencetest. Wild type tumor cells that express active UCP in the cell membranebut not in the mitochondrial or lysosomal membranes are not adherent totissue culture flasks having a negative charge but are adherent topoly-lysine which is positively charged. Cells which do not express UCPin the plasma membrane but do express mitochondrial or lysosomal UCP,such as melanoma cells, do not adhere to poly-lysine but adhere well tonegatively charged tissue culture dishes. Thus the adherence assay canbe used to detect the presence of active plasma membrane UCP orlysosomal UCP.

The invention also relates to the discovery that UCP is expressed in thelysosomal membrane. This finding has important implications for theregulation of many physiological processes including, antigenpresentation, respiratory burst, cholesterol to trafficking andinflammatory disease.

Antigen presentation is a complex process involving a pathway ofintracellular trafficking and peptide loading of MHC class II moleculesin the lysosome. Manipulation of elements involved in this pathway canresult in regulation of immune system function. One important element inthe lysosomal processing of antigen is pH. The lysosome must maintain anacidic pH in order to properly process antigen and ultimately presentantigen in the context of MHC class II. It has been discovered thatlysosomal pH can be manipulated by manipulating lysosomal UCP expressionand activity. If active UCP is expressed in the lysosome protons aredissipated and the pH is altered, thus preventing antigen presentation.If the expression or activity of the UCP is inhibited the lysosome candevelop an acidic pH to promote antigen presentation.

Thus the invention in some aspects relates to methods of inhibitinglysosomal UCP activity. It is particularly useful to inhibit lysosomalUCP for the prevention and treatment of infections. Thus, the inventionrelates to the prevention and treatment of infectious disease byinhibiting lysosomal UCP. Although not bound by any mechanism, Applicantbelieves that the inhibition of lysosomal UCP activity causes twophysiological effects which leads to the prevention and treatment ofinfections. Firstly, the inhibition of lysosomal UCP promotes thedevelopment of an acidic intra-lysosomal environment, which promotesantigen presentation. When antigen presenting cells (APCs) are exposedto an antigen of an infectious organism under conditions in whichlysosomal UCP activity is inhibited the antigen can be processed andpresented on the cell surface. The immune system can then mount aneffective antigen specific immune response against the antigen, thusproviding the host with a prophylactic or therapeutic immune response.

Additionally, inhibition of lysosomal UCP can promote respiratory burst,which is useful for treating intracellular pathogens. Respiratory burstis a process in which cells, e.g., neutrophils, macrophage undergo alarge burst in respiration and convert oxygen to toxic oxygenmetabolites such as the superoxide anion. This class of metabolites,also referred to as reactive oxygen species (ROS) include but are notlimited to hydrogen peroxide, superoxide anion, hypochlorous acid,peroxynitritesinglet oxygen, and hydroxyl radical. ROS are believed todamage microbes by reacting with proteins, nucleic acids, and membranelipids, and preventing these molecules from functioning. These compoundsare generated in large quantities during respiratory burst and functioneither intracellularly or are released into the extracellular space.

Thus, in one aspect, the present invention provides a method forpreventing or treating an infectious disease or cancer in a subjecthaving or at risk of developing an infectious disease or cancer byadministering a lysosomal UCP inhibitor to a cell of the subject in anamount effective to prevent lysosomal UCP activity. A “lysosomal UCPinhibitor” is any molecular species that prevents UCP activity in thelysosome. The lysosomal UCP inhibitor may function by preventing theactivity of an expressed UCP, preventing the transcription of alysosomal UCP gene, preventing the processing or translation of alysosomal UCP mRNA or preventing the processing, trafficking, oractivity of a lysosomal UCP protein when administered in vivo or invitro to a mammalian cell which is otherwise competent to express activelysosomal UCP. Thus, for example, lysosomal UCP inhibitors includelysosomal targeted nucleotides, nucleotide analogs, and bindingpeptides, repressors which prevent induction and/or transcription of thelysosomal UCP gene, antisense sequences which selectively bind tolysosomal UCP DNA or RNA sequences and which prevent the transcriptionor translation of the lysosomal UCP gene, competitive andnon-competitive inhibitors of the activity of the lysosomal UCP protein.In some embodiments of the invention the lysosomal UCP inhibitor is alysosomal UCP binding molecule or a lysosomal UCP antisense molecule.UCP binding proteins are those described above such as antibodies,including fragments of antibodies, such as Fc. These peptides aretargeted to the lysosomal membranes in order to selectively bind to andinhibit the activity of lysosomal UCP. Other types of inhibitors includeribozymes which interfere with the transcription, processing, ortranslation of lysosomal UCP mRNA. In other embodiments the UCPinhibitor is a nucleotide or nucleotide analog targeted to the lysosome.These nucleotides and analogs are those described above, such as ATP.

Another preferred lysosomal UCP inhibitor is tunicamycin. Tunicamycinpromotes intracellular trafficking of the lysosomal UCP from theintracellular location to the plasma membrane. When cells areadministered tunicamycin the UCP is selectively targeted away from thelysosome, preventing respiratory burst and promoting antigenpresentation. If tunicamycin therapy is combined with a plasma membranetargeted UCP to inhibitor the cells are killed.

The terms “prevent” and “preventing” as used herein refer to inhibitingcompletely or partially the expression or activity of a UCP molecule, aswell as inhibiting an increase in the expression or activity of a UCPmolecule or inhibiting completely or partially antigen presentation orrespiratory burst or inhibiting an increase in antigen presentation orrespiratory burst.

Thus, in some aspects, the invention encompasses a method forstimulating an MHC class II immune response by administering to a cell alysosomal UCP inhibitor in an amount effective to prevent lysosomal UCPactivity. The method is performed in order to enhance antigenic peptideloading in MHC class II complexes, and for promoting specific antigenimmune responses. Therefore, in one aspect, the present inventionprovides methods for promoting antigen-specific immune responses. TheUCP inhibitor can be administered in conjunction with a specificantigen, such that that specific antigen is preferentially loaded intothe MHC class II molecules. These methods are useful for both in vitroand in vivo antigen loading.

When used with mammalian cells in vitro, such methods have utility forloading of specific antigens within the MHC molecules. Cells withspecific antigen loading in class II molecules have utility in a varietyof analytical and diagnostic assays. These cells are also useful astherapeutic agents. For instance, the cells can be used in culture tostudy immune responses or to screen the effect of putative drugs oninhibiting or promoting antigen-specific immune responses. Additionally,the cells could be administered to a mammalian subject to promote anantigen-specific T cell response. When administered to a subject, theclass II MHC/antigen complexes on the surface of the cell would interactwith endogenous T cells, inducing an immune cascade, and thus producingan antigen-specific immune response. In preferred embodiments, the cellsmanipulated in vitro have been isolated from the same subject ex vivo.

The UCP inhibitors are also useful for treating a mammalian subject invivo to induce an antigen-specific immune response. It is useful toproduce antigen-specific immune responses against any foreign antigenwhether it is capable of causing a pathological state or any damage toits mammalian host. The terms “foreign antigen” or “antigen” are usedsynonymously to refer to a molecule capable of provoking an immuneresponse in a host, wherein the antigen is not a self-antigen, asdefined above. Thus, the term antigen or foreign antigen specificallyexcludes self-antigens. Self-antigens are used herein to refer to thepeptide-antigens of autoimmune disorders. An immune response against theself-antigen results in an autoimmune disorder. The term self-antigendoes not include, however, antigens such as cancer antigens, which arerecognized by the host as foreign and which are not associated withautoimmune disease. Thus, the term antigen specifically excludesself-antigens and broadly includes any type of molecule (e.g. associatedwith a host or foreign cell) which is recognized by a host immune systemas being foreign. Antigens include, but are not limited to, cancerantigens and microbial antigens and may be composed of cells, cellextracts, polysaccharides, polysaccharide conjugates, lipids,glycolipids, carbohydrates, peptides, proteins, viruses, viral extracts,etc.

A “cancer antigen”, as used herein, is a compound which is associatedwith a tumor or cancer cell surface and which is capable of provoking animmune response when expressed on the surface of an antigen-presentingcell in the context of a class II MHC molecule. Cancers or tumorsinclude those described above.

Cancer antigens include but are not limited to Melan-A/MART-1,Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein(ADAbp), cyclophilin b, Colorectal associated antigen(CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenicepitopes CAP-1 and CAP-2, etv6, am11, Prostate Specific Antigen (PSA)and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family oftumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-Ail, MAGE-A12,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-family of tumor antigens(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53,MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin,α-catenin, β-catenin and γ-catenin, p120ctn, gp100^(Pmel117), PRAME,NY-ESO-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, cdc27, adenomatous polyposis coliprotein (APC), fodrin, PIA, Connexin 37, Ig-idiotype, p15, gp75, GM2 andGD2 gangliosides, viral products such as human papilloma virus proteins,Smad family of tumor antigens, lmp-1, EBV-encoded nuclear antigen(EBNA)-1, or c-erbB-2.

In some embodiments, cancers or tumors escaping immune recognition andtumor-antigens associated with such tumors (but not exclusively),include acute lymphoblastic leukemia (etv6; am11; cyclophilin b), B celllymphoma (Ig-idiotype), glioma (E-cadherin; α-catenin; β-catenin;γ-catenin; p120ctn), bladder cancer (p21ras), billiary cancer (p21ras),breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53;p21ras), colon carcinoma (p21ras; HER2/neu; c-erbB-2; MUC family),colorectal cancer (Colorectal associated antigen (CRC)-0017-1A/GA733;APC), choriocarcinoma (CEA), epithelial cell-cancer (cyclophilin b),gastric cancer (HER2/neu; c-erbB-2; ga733 glycoprotein), hepatocellularcancer (α-fetoprotein), hodgkins lymphoma (lmp-1; EBNA-1), lung cancer(CEA; MAGE-3; NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b),melanoma (p15 protein, gp75, oncofetal antigen, GM2 and GD2gangliosides), myeloma (MUC family; p21ras), non-small cell lungcarcinoma (HER2/neu; c-erbB-2), nasopharyngeal cancer (lmp-1; EBNA-1),ovarian cancer cancer (MUC family; HER2/neu; c-erbB-2), prostate cancer(Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1,PSA-2, and PSA-3; PSMA; HER2/neu; c-erbB-2), pancreatic cancer (p21ras;MUC family; HER2/neu; c-erbB-2; ga733 glycoprotein), renal (HER2/neu;c-erbB-2), squamous cell cancers of cervix and esophagus (viral productssuch as human papilloma virus proteins), testicular cancer (NY-ESO-1), Tcell leukemia (HTLV-1 epitopes), and melanoma (Melan-A/MART-1; cdc27;MAGE-3; p21ras; gp100^(Pmel117)). These antigens are also usefulaccording to the invention.

For examples of tumor antigens which bind to either or both MHC class Iand MHC class II molecules, see the following references: Coulie, StemCells 13:393-403, 1995; Traversari et al., J. Exp. Med. 176:1453-1457,1992; Chaux et al., J. Immunol. 163:2928-2936, 1999; Fujie et al., Int.J. Cancer 80:169-172, 1999; Tanzarella et al., Cancer Res. 59:2668-2674,1999; van der Bruggen et al., Eur. J. Immunol. 24:2134-2140, 1994; Chauxet al., J. Exp. Med. 189:767-778, 1999; Kawashima et al, Hum. Immunol.59:1-14, 1998; Tahara et al., Clin. Cancer Res. 5:2236-2241, 1999;Gaugler et al., J. Exp. Med. 179:921-930, 1994; van der Bruggen et al.,Eur. J. Immunol. 24:3038-3043, 1994; Tanaka et al., Cancer Res.57:4465-4468, 1997; Oiso et al., Int. J. Cancer 81:387-394, 1999; Hermanet al., Immunogenetics 43:377-383, 1996; Manici et al., J. Exp. Med.189:871-876, 1999; Duffour et al., Eur. J. Immunol. 29:3329-3337, 1999;Zorn et al., Eur. J. Immunol. 29:602-607, 1999; Huang et al., J.Immunol. 162:6849-6854, 1999; Boël et al., Immunity 2:167-175, 1995; Vanden Eynde et al., J. Exp. Med. 182:689-698, 1995; De Backer et al.,Cancer Res. 59:3157-3165, 1999; Jäger et al., J. Exp. Med. 187:265-270,1998; Wang et al., J. Immunol. 161:3596-3606, 1998; Aarnoudse et al.,Int. J. Cancer 82:442-448, 1999; Guilloux et al., J. Exp. Med.183:1173-1183, 1996; Lupetti et al., J. Exp. Med. 188:1005-1016, 1998;Wölfel et al., Eur. J. Immunol. 24:759-764, 1994; Skipper et al., J.Exp. Med. 183:527-534, 1996; Kang et al., J. Immunol. 155:1343-1348,1995; Morel et al., Int. J. Cancer 83:755-759, 1999; Brichard et al.,Eur. J. Immunol. 26:224-230, 1996; Kittlesen et al., J. Immunol.160:2099-2106, 1998; Kawakami et al., J. Immunol. 161:6985-6992, 1998;Topalian et al., J. Exp. Med. 183:1965-1971, 1996; Kobayashi et al.,Cancer Research 58:296-301, 1998; Kawakami et al., J. Immunol.154:3961-3968, 1995; Tsai et al., J. Immunol. 158:1796-1802, 1997; Coxet al., Science 264:716-719, 1994; Kawakami et al., Proc. Natl. Acad.Sci. USA 91:6458-6462, 1994; Skipper et al., J. Immunol. 157:5027-5033,1996; Robbins et al., J. Immunol. 159:303-308, 1997; Castelli et al, J.Immunol. 162:1739-1748, 1999; Kawakami et al., J. Exp. Med. 180:347-352,1994; Castelli et al., J. Exp. Med. 181:363-368, 1995; Schneider et al.,Int. J. Cancer 75:451-458, 1998; Wang et al., J. Exp. Med.183:1131-1140, 1996; Wang et al., J. Exp. Med. 184:2207-2216, 1996;Parkhurst et al., Cancer Research 58:4895-4901, 1998; Tsang et al., J.Natl Cancer Inst 87:982-990, 1995; Correale et al., J Natl Cancer Inst89:293-300, 1997; Coulie et al., Proc. Natl. Acad. Sci. USA92:7976-7980, 1995; Wölfel et al., Science 269:1281-1284, 1995; Robbinset al., J. Exp. Med. 183:1185-1192, 1996; Brändle et al., J. Exp. Med.183:2501-2508, 1996; ten Bosch et al., Blood 88:3522-3527, 1996;Mandruzzato et al., J. Exp. Med. 186:785-793, 1997; Gueguen et al., J.Immunol. 160:6188-6194, 1998; Gjertsen et al., Int. J. Cancer72:784-790, 1997; Gaudin et al., J. Immunol. 162:1730-1738, 1999; Chiariet al., Cancer Res. 59:5785-5792, 1999; Hogan et al., Cancer Res.58:5144-5150, 1998; Pieper et al., J. Exp. Med. 189:757-765, 1999; Wanget al., Science 284:1351-1354, 1999; Fisk et al., J. Exp. Med.181:2109-2117, 1995; Brossart et al., Cancer Res. 58:732-736, 1998;ROpke et al., Proc. Natl. Acad. Sci. USA 93:14704-14707, 1996; Ikeda etal., Immunity 6:199-208, 1997; Ronsin et al., J. Immunol. 163:483-490,1999; Vonderheide et al., Immunity 10:673-679, 1999. These antigens aswell as others are disclosed in PCT Application PCT/US98/18601.

In other aspects, the antigen is a microbial antigen and the methods ofthe invention are useful for treating or preventing infectious disease.An infectious disease, as used herein, is a disease arising from thepresence of a foreign microorganism in the body. A microbial antigen, asused herein, is an antigen of a microorganism and, includes but it notlimited to, infectious virus, infectious bacteria, and infectious fungi.

Examples of infectious virus include but are not limited to:Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and otherisolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitisA virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g.vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

Examples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Other infectious organisms(i.e., protists) include: Plasmodium such as Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax andToxoplasma gondii.

The lysosomal UCP inhibitors are also useful for treating disorderssusceptible to respiratory burst Inhibition of lysosomal UCP allowscells to produce reactive oxygen species in order to kill intracellularpathogens. Thus, in some aspects the invention relates to a method oftreating a subject infected with an intracellular pathogen. In oneembodiment the lysosomal UCP inhibitor is tunicamycin.

Microorganisms can be classified based on whether they are intracellularor extracellular. An intracellular pathogen, as used herein, is apathogen whose entire life cycle is intracellular or a pathogen whichhas an obligate intracellular existence at a critical stage in theirlife cycles. Examples of human intracellular parasites includeLeishmania spp., Plasmodium spp., Trypanosoma cruzi, Toxoplasma gondii,Babesia spp., and Trichinella spiralis. Other intracellular pathogenssuch as bacteria are well known in the art. Parasites which are mainlyextracellular but with an obligate intracellular existence at a criticalstage in their life cycles are referred to herein as “obligateintracellular parasites”. These parasites may exist most of their livesor only a small portion of their lives in an extracellular environment,but they all have at lest one obligate intracellular stage in their lifecycles. This latter category of parasites includes Trypanosomarhodesiense and Trypanosoma gambiense, Isospora spp., Cryptosporidiumspp, Eimeria spp., Neospora spp., Sarcocystis spp., and Schistosoma spp.The methods of the invention are useful for treating infection resultingfrom both intracellular pathogens and obligate intracellular pathogenswhich have at least in one stage of their life cycle that isintracellular. Blood-borne and/or tissues parasites include Plasmodiumspp., Babesia microti, Babesia divergens, Leishmania tropica, Leishmaniaspp., Leishmania braziliensis, Leishmania donovani, Trypanosomagambiense and Trypanosoma rhodesiense (African sleeping sickness),Trypanosoma cruzi (Chagas' disease), and Toxoplasma gondii.

Activators of lysosomal UCP are useful for raising the lysosomal pH andpreventing antigen presentation and respiratory burst as well as forpreventing trafficking and preventing and treating inflammatory disease.When used in vivo, methods of the invention in some aspects are usefulfor treating or preventing disorders associated with a specificantigenic immune response. Thus, in some embodiments of the invention,the methods are used to treat mammals at risk of, or afflicted with,autoimmune disease. Autoimmune disease is a disorder in which the host'simmune response is defective and results in the production of a specificimmune response against the individual's own antigens or components. Inan autoimmune disease, an individual's own antibodies react with hosttissue or in which immune effector T cells are autoreactive toendogenous self peptides and cause destruction of tissue. It is wellestablished that MHC class II alleles act as major genetic elements insusceptibility to a variety of autoimmune diseases. The structuresrecognized by T cells, the cells that cause autoimmunity, are complexescomprised of class II MHC molecules and antigenic peptides. When the Tcells react with the host's class II MHC molecules-peptide complexesderived from a host's own gene products, autoimmune disease can result.If these class II MHC/peptide complexes are inhibited from being formed,the autoimmune response is reduced or suppressed, and thus is inhibitedaccording to the invention. The peptide-antigen of autoimmune disordersare self-antigens. Any autoimmune disease in which class II MHC/peptidecomplexes play a role may be treated according to the methods of thepresent invention. Such autoimmune diseases include, but are not limitedto, juvenile-onset diabetes (insulin-dependent), multiple sclerosis,pemphigus vulgaris, Graves disease, myasthenia gravis, systemic lupuserythematosus (SLE), celiac disease rheumatoid arthritis, andHashimoto's thyroiditis.

The methods of the invention are also useful for treating mammals atrisk of, or afflicted with, allergic responses. An “allergic response”as used herein is a disorder in which the host's immune response to aparticular antigen is unnecessary or disproportionate, resulting inpathology. An allergic response may occur, in part, because a T cellrecognizes a particular class II MHC/peptide complex and triggers acascade of immune response. If the class II MHC/peptide complex isinhibited from being formed, the allergic response is reduced orsuppressed. Any allergic response in which class II MHC/peptidecomplexes play a role may be treated according to the methods of thepresent invention. Allergies arising from an allergic response include,but are not limited to, allergies to pollen, ragweed, shellfish,domestic animals, (e.g., cats and dogs), B-venom, and the like. A subsetof allergic responses produce asthma. Allergic asthmatic responses arealso included within the definition of the term “allergic response”. Itis particularly desirable to treat severe or life-threatening allergicresponses, such as those arising during asthmatic attacks oranaphylactic shock, according to the methods of the invention.

The methods of the invention are also useful for treating mammals whichhave undergone or about to undergo, an organ transplant or tissue graft.In tissue transplantation (e.g., kidney, lung, liver, heart) or skingrafting, when there is a mismatch between the class II MHC genotypes(HLA types) of the donor and recipient, there may be a severe“allogeneic immune response” against the donor tissues which resultsfrom the presence of non-self or allogeneic class II MHC moleculespresenting antigenic peptides on the surface of donor cells.

The activation of lysosomal UCP will inhibit the formation of class IIMHC/antigenic peptide complexes, resulting in a suppression ormitigation of tissue rejection. The UCP activator may be used alone orin conjunction with other therapeutic agents, e.g., as an adjunct tocyclosporin A and/or antilymphocyte gammaglobulin to promote graftsurvival. The administration of the UCP activator with or without othertherapeutic agents may be performed before or after the surgery bysystemic administration or may be perfused directly into the donor organor tissue prior to or subsequent to transplantation or grafting.

UCP activators also include UCP peptides which can be delivered to orexpressed to within a lysosome of a cell such as an APC. APC is usedherein to specifically refer to immune cells that can express MHC classII/antigen on the surface. In some cases, this class of compoundsincludes peptides which are complete UCP proteins or functionally activefragments which are targeted to the lysosome. In other embodiments, thisclass of compounds includes nucleic acids which produce a lysosomaltargeted UCP. Other preferred lysosomal UCP activators are lysosomaltargeted omega-3 fatty acids, such as oleic acid, palmitic acid andmyrisitate.

In some aspects the methods of the invention include methods forpreventing cholesterol trafficking. The method is accomplished byadministering a lysosomal UCP activator to a subject to prevent lipidaccumulation. Although applicants are not bound by the mechanism it isbelieved that lysosomal UCP activators prevent cholesterol traffickingby increasing UCP activity and thus promoting use of fatty acids forfuel. Inactive lysosomal UCP results in lipid accumulation leading tocholesterol accumulation.

The lysosomal UCP activators are also useful for preventing inflammatorydisease. Inflammatory disease is characterized by inflammationassociated with neutrophil accumulation and activation due to excessiveneutrophil stimulation. While not intending to be bound by anyparticular theory, it is believed that when lysosomal UCP is inactiveneutrophils undergo respiratory burst and actively accumulate at a siteof injury, where they release toxic factors and damage surroundingtissue. When UCP is active in the lysosome neutrophil activation isinhibited and the toxic factors are not released into the surroundingtissues. When the inflammatory disease is an acute stroke a tissue whichis often damaged by neutrophil stimulation is the brain. As the activeneutrophils accumulate in the brain an infarct may develop.

An “inflammatory disease or condition” as used herein refers to anycondition characterized by local inflammation at a site of injury orinfection and includes autoimmune diseases, certain forms of infectiousinflammatory states, undesirable neutrophil activity characteristic oforgan transplants or other implants and virtually any other conditioncharacterized by unwanted neutrophil activation. These conditionsinclude but are not limited to meningitis, cerebral edema, arthritis,nephritis, adult respiratory distress syndrome, pancreatitis, myositis,neuritis, connective tissue diseases, phlebitis, arteritis, vasculitis,allergy, anaphylaxis, ehrlichiosis, gout, organ transplants and/orulcerative colitis.

The invention also includes compositions of the above described agents.One composition of the invention includes an UCP associated with aplasma membrane targeting molecule. Each of these compounds is describedabove in more detail. Another composition of the invention includes anUCP inhibitor associated with a plasma membrane targeting molecule.

Each of the compositions of the invention may optionally be associatedwith a delivery system or vector. In its broadest sense, a “vector” isany vehicle capable of facilitating: (1) delivery of a composition to atarget cell or (2) uptake of a composition by a target cell, if uptakeis important. Optionally, a “targeting ligand” (in addition to or thesame as the plasma membrane targeting molecule) can be attached to thevector to selectively deliver the vector to a cell which expresses onits surface the cognate receptor for the targeting ligand. In thismanner, the vector (containing a composition of the invention) can beselectively delivered to a cell in, e.g., a tumor. In general, thevectors useful in the invention are divided into two classes: colloidaldispersion systems and biological vectors.

As used herein, a “colloidal dispersion system” refers to a natural orsynthetic molecule, other than those derived from bacteriological orviral sources, capable of delivering to and releasing the composition ina subject. Colloidal dispersion systems include macromolecularcomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvessels (LUV), which range in size from 0.2-4.0μ can encapsulate largemacromolecules within the aqueous interior and these macromolecules canbe delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci., 6:77 (1981)).

Lipid formulations for transfection are commercially available fromQIAGEN, for example as EFFECTENE™ (a non-liposomal lipid with a specialDNA condensing enhancer) and SUPER-FECTT™ (a novel acting dendrimerictechnology) as well as Gibco BRL, for example, as LIPOFECTIN™ andLIPOFECTACE™, which are formed of cationic lipids such as N-[1-(2, 3dioleyloxy)-propyl]-N,N, N-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Methods for makingliposomes are well known in the art and have been described in manypublications. Liposomes were described in a review article byGregoriadis, G., Trends in Biotechnology 3:235-241 (1985), which ishereby incorporated by reference.

In one particular embodiment, the preferred vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Internationalapplication no. PCT/US/03307 (Publication No. WO 95/24929, entitled“Polymeric Gene Delivery System”, claiming priority to U.S. patentapplication serial no. 213,668, filed Mar. 15, 1994). PCT/US/0307describes a biocompatible, preferably biodegradable polymeric matrix forcontaining an exogenous gene under the control of an appropriatepromotor. The polymeric matrix is used to achieve sustained release ofthe exogenous gene in the patient. In accordance with the instantinvention, the compositions of the invention described herein areencapsulated or dispersed within the biocompatible, preferablybiodegradable polymeric matrix disclosed in PCT/US/03307.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein the composition is dispersed throughout asolid polymeric matrix) or a microcapsule (wherein the composition isstored in the core of a polymeric shell). Other forms of the polymericmatrix for containing the composition include films, coatings, gels,implants, and stents. The size and composition of the polymeric matrixdevice is selected to result in favorable release kinetics in the tissueinto which the matrix is introduced. The size of the polymeric matrixfurther is selected according to the method of delivery which is to beused, typically injection into a tissue or administration of asuspension by aerosol into the nasal and/or pulmonary areas. Preferablywhen an aerosol route is used the polymeric matrix and composition areencompassed in a surfactant vehicle. The polymeric matrix compositioncan be selected to have both favorable degradation rates and also to beformed of a material which is bioadhesive, to further increase theeffectiveness of transfer when the matrix is administered to a nasaland/or pulmonary surface that has sustained an injury. The matrixcomposition also can be selected not to degrade, but rather, to releaseby diffusion over an extended period of time.

In another embodiment the chemical/physical vector is a biocompatiblemicrosphere that is suitable for oral delivery. Such microspheres aredisclosed in Chickering et al., Biotech. And Bioeng., (1996) 52:96-101and Mathiowitz et al., Nature, (1997) 386:410-414.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the compositions of the invention of the invention to thesubject. Biodegradable matrices are preferred. Such polymers may benatural or synthetic polymers. Synthetic polymers are preferred. Thepolymer is selected based on the period of time over which release isdesired, generally in the order of a few hours to a year or longer.Typically, release over a period ranging from between a few hours andthree to twelve months is most desirable. The polymer optionally is inthe form of a hydrogel that can absorb up to about 90% of its weight inwater and further, optionally is cross-linked with multi-valent ions orother polymers.

In general, the compositions of the invention are delivered using abioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene, polyvinylpyrrolidone, and polymers of lactic acidand glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid),poly(valeric acid), and poly(lactide-cocaprolactone), and naturalpolymers such as alginate and other polysaccharides including dextranand cellulose, collagen, chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), albumin and other hydrophilic proteins, zeinand other prolamines and hydrophobic proteins, copolymers and mixturesthereof. In general, these materials degrade either by enzymatichydrolysis or exposure to water in vivo, by surface or bulk erosion.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, (1993) 26:581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

It is envisioned that the UCP or UCP inhibitor may be delivered to thesubject in a biological vector which is a nucleic acid molecule whichencodes for the UCP or UCP inhibitor such that the UCP or UCP inhibitormust be expressed in vivo. The nucleic acid encoding the UCP or UCPinhibitor is operatively linked to a gene expression sequence whichdirects the expression of the UCP or UCP inhibitor nucleic acid within aeukaryotic cell. The “gene expression sequence” is any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient transcription andtranslation of the UCP or UCP inhibitor nucleic acid to which it isoperatively linked. The gene expression sequence may, for example, be amammalian or viral promoter, such as a constitutive or induciblepromoter. Constitutive mammalian promoters include, but are not limitedto, the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, β-actinpromoter and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the simian virus, papilloma virus, adenovirus, humanimmunodeficiency virus (HIV), rous sarcoma virus, cytomegalovirus, thelong terminal repeats (LTR) of moloney leukemia virus and otherretroviruses, and the thymidine kinase promoter of herpes simplex virus.Other constitutive promoters are known to those of ordinary skill in theart. The promoters useful as gene expression sequences of the inventionalso include inducible promoters. Inducible promoters are expressed inthe presence of an inducing agent. For example, the metallothioneinpromoter is induced to promote transcription and translation in thepresence of certain metal ions. Other inducible promoters are known tothose of ordinary skill in the art.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined UCP or UCP inhibitor nucleic acid. The gene expression sequencesoptionally include enhancer sequences or upstream activator sequences asdesired.

The UCP or UCP inhibitor nucleic acid is operatively linked to the geneexpression sequence. As used herein, the UCP or UCP inhibitor nucleicacid sequence and the gene expression sequence are said to be “operablylinked” when they are covalently linked in such a way as to place theexpression or transcription and/or translation of the UCP or UCPinhibitor coding sequence under the influence or control of the geneexpression sequence. Two DNA sequences are said to be operably linked ifinduction of a promoter in the 5′ gene expression sequence results inthe transcription of the UCP or UCP inhibitor sequence and if the natureof the linkage between the two DNA sequences does not (1) result in theintroduction of a frame-shift mutation, (2) interfere with the abilityof the promoter region to direct the transcription of the UCP or UCPinhibitor sequence, or (3) interfere with the ability of thecorresponding RNA transcript to be translated into a protein. Thus, agene expression sequence would be operably linked to an UCP or UCPinhibitor nucleic acid sequence if the gene expression sequence werecapable of effecting transcription of that UCP or UCP inhibitor nucleicacid sequence such that the resulting transcript is translated into thedesired protein or polypeptide.

The UCP or UCP inhibitor nucleic acid of the invention may be deliveredto the cell alone or in association with a vector. In its broadestsense, a “vector” is any vehicle capable of facilitating the transfer ofthe UCP or UCP inhibitor nucleic acid to the appropriate cells so thatthe UCP or UCP inhibitor can be expressed on the plasma membrane orwithin the cell respectively. Preferably, the vector transports thenucleic acid to the cells with reduced degradation relative to theextent of degradation that would result in the absence of the vector.The vector optionally includes the above-described gene expressionsequence to enhance expression of the UCP or UCP inhibitor nucleic acid.In general, the vectors useful in the invention include, but are notlimited to, plasmids, phagemids, viruses, other vehicles derived fromviral or bacterial sources that have been manipulated by the insertionor incorporation of the UCP or UCP inhibitor nucleic acid sequences.Viral vectors are a preferred type of vector and include, but are notlimited to nucleic acid sequences from the following viruses:retrovirus, such as moloney murine leukemia virus, harvey murine sarcomavirus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus,adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barrviruses; papilloma viruses; herpes virus; vaccinia virus; polio virus;and RNA virus such as a retrovirus. One can readily employ other vectorsnot named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses, the life cycle ofwhich involves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell lined withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., “Gene Transfer and Expression, A Laboratory Manual,” W.H.Freeman C.O., New York (1990) and Murry, E. J. Ed. “Methods in MolecularBiology,” vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

A preferred virus for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus can beengineered to be replication-deficient and is capable of infecting awide range of cell types and species. It further has advantages such as,heat and lipid solvent stability; high transduction frequencies in cellsof diverse lineages, including hemopoietic cells; and lack ofsuperinfection inhibition thus allowing multiple series oftransductions. Reportedly, the adeno-associated virus can integrate intohuman cellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression characteristic of retroviral infection. In addition,wild-type adeno-associated virus infections have been followed in tissueculture for greater than 100 passages in the absence of selectivepressure, implying that the adeno-associated virus genomic integrationis a relatively stable event. The adeno-associated virus can alsofunction in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See e.g., Sambrook et al., “Molecular Cloning: A LaboratoryManual,” Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been found to be particularlyadvantageous for delivering genes to cells in vivo because of theirinability to replicate within and integrate into a host genome. Theseplasmids, however, having a promoter compatible with the host cell, canexpress a peptide from a gene operatively encoded within the plasmid.Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40,and pBlueScript. Other plasmids are well-known to those of ordinaryskill in the art. Additionally, plasmids may be custom designed usingrestriction enzymes and ligation reactions to remove and add specificfragments of DNA.

It has recently been discovered that gene carrying plasmids can bedelivered to the cells in vivo using bacteria. Modified forms ofbacteria such as Salmonella can be transfected with the plasmid and usedas delivery vehicles. The bacterial delivery vehicles can beadministered to a host subject orally or by other administration means.The bacteria deliver the plasmid to cells probably by passing throughthe gut barrier. High levels of expression have been established usingthis methodology.

Compaction agents also can be used alone, or in combination with, avector of the invention. A “compaction agent”, as used herein, refers toan agent, such as a histone, that neutralizes the negative charges onthe nucleic acid and thereby permits compaction of the nucleic acid intoa fine granule. Compaction of the nucleic acid facilitates the uptake ofthe nucleic acid by the target cell. The compaction agents can be usedalone, i.e., to deliver the compositions in a form that is moreefficiently taken up by the cell or, more preferably, in combinationwith one or more of the above-described vectors.

Other exemplary compositions that can be used to facilitate uptake by atarget cell of the compositions of the invention include calciumphosphate and other chemical mediators of intracellular transport,microinjection compositions, electroporation and homologousrecombination compositions (e.g., for integrating a composition of theinvention into a preselected location within the target cellchromosome).

The pharmaceutical preparations of the invention are administered tosubjects in effective amounts. An effective amount means that amountnecessary to delay the onset of, inhibit the progression of, haltaltogether the onset or progression of or diagnose the particularcondition being treated. In one embodiment the compositions are presentin an effective dose for treating a tumor. In another embodiment thecompositions are present in an effective dose for treating type IIdiabetes. In general, an effective amount for treating cancer and type Idiabetes will be that amount necessary to favorably affect mammaliancell proliferation in-situ. When administered to a subject, effectiveamounts will depend, of course, on the particular condition beingtreated; the severity of the condition; individual patient parametersincluding age, physical condition, size and weight; concurrenttreatment; frequency of treatment; and the mode of administration. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is preferredgenerally that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment.

Generally, doses of active compounds will be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses range of 50-500mg/kg will be suitable, in one or several administrations per day. Inthe event that a response in a subject is insufficient at the initialdoses applied, higher doses (or effectively higher doses by a different,more localized delivery route) may be employed to the extent thatpatient tolerance permits. Multiple doses per day are contemplated toachieve appropriate levels of compounds.

In other aspects the invention includes transgenic animals and cellstransfected with the UCP's. Additionally, complements of the UCP nucleicacids described above can be useful as anti-sense oligonucleotides,e.g., by delivering the anti-sense oligonucleotide to an animal toinduce a “knockout” phenotype. The administration of anti-sense RNAprobes to block gene expression is discussed in Lichtenstein, C., Nature333:801-802 (1988).

Alternatively, the UCP nucleic acids can be used to prepare a non-humantransgenic animal. A “transgenic animal” is an animal having cells thatcontain DNA which has been artificially inserted into a cell, which DNAbecomes part of the genome of the animal which develops from that cell.Preferred transgenic animals are primates, mice, rats, cows, pigs,horses, goats, sheep, dogs and cats. Animals suitable for transgenicexperiments can be obtained from standard commercial sources such asCharles River (Wilmington, Mass.), Taconic (Germantown, N.Y.), HarlanSprague Dawley (Indianapolis, Ind.), etc. Transgenic animals having aparticular property associated with a particular disease can be used tostudy the affects of a variety of drugs and treatment methods on thedisease, and thus serve as genetic models for the study of a number ofhuman diseases. The invention, therefore, contemplates the use of UCPknockout and transgenic animals as models for the study of proliferativedisorders.

A variety of methods are available for the production of transgenicanimals associated with this invention. DNA can be injected into thepronucleus of a fertilized egg before fusion of the male and femalepronuclei, or injected into the nucleus of an embryonic cell (e.g., thenucleus of a two-cell embryo) following the initiation of cell division.See e.g., Brinster et al., Proc. Nat. Acad. Sci. USA, 82: 4438 (1985);Brinster et al., cell 27: 223 (1981); Costantini et al., Nature 294: 982(1981); Harpers et al., Nature 293: 540 (1981); Wagner et al., Proc.Nat. Acad. Sci. USA 78:5016 (1981); Gordon et al., Proc. Nat. Acad. Sci.USA 73: 1260 (1976). The fertilized egg is then implanted into theuterus of the recipient female and allowed to develop into an animal.

An alternative method for producing transgenic animals involves theincorporation of the desired gene sequence into a virus which is capableof affecting the cells of a host animal. See e.g., Elbrecht et al.,Molec. Cell. Biol. 7: 1276 (1987); Lacey et al., Nature 322: 609 (1986);Leopol et al., Cell 51: 885 (1987). Embryos can be infected withviruses, especially retroviruses, modified to carry the nucleotidesequences which encode UCP proteins or sequences which disrupt thenative UCP gene to produce a knockout animal.

Another method for producing transgenic animals involves the injectionof pluripotent embryonic stem cells into a blastocyst of a developingembryo. Pluripotent stem cells derived from the inner cell mass of theembryo and stabilized in culture can be manipulated in culture toincorporate nucleotide sequences of the invention. A transgenic animalcan be produced from such cells through implantation into a blastocystthat is implanted into a foster mother and allowed to come to term. Seee.g., Robertson et al., Cold Spring Harbor Conference Cell Proliferation10: 647 (1983); Bradley et al., Nature 309: 255 (1984); Wagner et al.,Cold Spring Harbor Symposium Quantitative Biology 50: 691 (1985).

The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout, Experientia, 47: 897-905 (1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No., 4,945,050 (Sandford et al., Jul. 30, 1990).

By way of example only, to prepare a transgenic mouse, female mice areinduced to superovulate. Females are placed with males, and the matedfemales are sacrificed by CO₂ asphyxiation or cervical dislocation andembryos are recovered from excised oviducts. Surrounding cumulus cellsare removed. Pronuclear embryos are then washed and stored until thetime of injection. Randomly cycling adult female mice are paired withvasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice. See Hammer etal., Cell 63:1099-1112 (1990).

Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as electroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art. See, for example, Teratocarcinomasand Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,IRL Press (1987).

In cases involving random gene integration, a clone containing thesequence(s) of the invention is co-transfected with a gene encodingresistance. Alternatively, the gene encoding neomycin resistance isphysically linked to the sequence(s) of the invention. Transfection andisolation of desired clones are carried out by any one of severalmethods well known to those of ordinary skill in the art (E. J.Robertson, supra).

DNA molecules introduced into ES cells can also be integrated into thechromosome through the process of homologous recombination. Capecchi,Science, 244: 1288-1292 (1989). Methods for positive selection of therecombination event (e.g., neo resistance) and dual positive-negativeselection (e.g., neo resistance and gangcyclovir resistance) and thesubsequent identification of the desired clones by PCR have beendescribed by Capecchi, supra and Joyner et al., Nature, 338: 153-156(1989). The final phase of the procedure is to inject targeted ES cellsinto blastocysts and to transfer the blastocysts into pseudopregnantfemales. The resulting chimeric animals are bred and the offspring areanalyzed by Southern blotting to identify individuals that carry thetransgene.

Procedures for the production of non-rodent mammals and other animalshave been discussed by others. See Houdebine and Chourrout, supra;Pursel et al., Science 244: 1281-1288 (1989); and Simms et al.,Bio/Technology, 6: 179-183 (1988).

Inactivation or replacement of the endogenous UCP genes can be achievedby a homologous recombination system using embryonic stem cells. Theresultant transgenic non-human mammals having a knockout characteristicmay be used as a model for disorders involving abnormally low levels ofproliferation. Resistant tumor cells which do not express UCPs maydevelop as a result of the inability to express UCP on the plasmamembrane. A variety of therapeutic drugs can be administered to thephenotypically chemotherapeutic resistant animals to determine theaffect of the therapeutic drugs on nerve cell differentiation. In thismanner, therapeutic drugs which are useful for preventing or reducingthese disorders can be identified.

Additionally, a normal or mutant version of UCP can be inserted into themouse germ line to produce transgenic animals which constitutively orinducible express the normal or mutant form of UCP in the plasmamembrane. These animals are useful in studies to define the role andfunction of UCP in cells and to observe disorders associated withabnormally high proliferation, such as tumors or autoimmune disease.

The UCP inhibitors and activators described herein are commerciallyavailable compounds, are derived from commercially available compoundsor are synthesized de novo using routine chemical synthetic proceduresknown to those of ordinary skill in the art and/or described herein.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts. As used herein, the compositions of theinvention may include various salts.

The compositions of the invention may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration into a human or other animal. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being co-mingled with the molecules of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the compositions of theinvention, which is preferably isotonic with the blood of the recipient.This aqueous preparation may be formulated according to known methodsusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation also may be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administrations canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

A variety of administration routes are available. The particular modeselected will depend of course, upon the particular drug selected, theseverity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes are not particularly suitable for long-term therapyand prophylaxis. They could, however, be preferred in emergencysituations. Oral administration will be preferred for prophylactictreatment because of the convenience to the patient as well as thedosing schedule.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing thecompositions of the invention into association with a carrier whichconstitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing thecompositions of the invention into association with a liquid carrier, afinely divided solid carrier, or both, and then, if necessary, shapingthe product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the compositions of the invention. Othercompositions include suspensions in aqueous liquids or non-aqueousliquids such as a syrup, elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions of the invention described above,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are: lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono- di- and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the compositions of the invention is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) difusional systemsin which an active component permeates at a controlled rate from apolymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480. Inaddition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

The inhibitors and activators of the invention can be administered byany method which allows the inhibitor or activator to reach the targetcells, e.g., class II MHC antigen-presenting cells. These methodsinclude, e.g., injection, infusion, deposition, implantation, anal orvaginal supposition, oral ingestion, inhalation, topical administration,or any other method of administration where access to the target cell bythe inhibitor is obtained. In some embodiments, topical administrationis preferred, due to the high concentration of APCs in the skin. Onemethod for accomplishing topical administration includes transdermaladministration, such as iontophoresis. Iontophoretic transmission can beaccomplished by using commercially-available patches which deliver acompound continuously through unbroken skin for periods of hours to daysto weeks, depending on the particular patch. This method allows for thecontrolled delivery of the inhibitors or activators through the skin inrelatively high concentrations. One example of an iontophoretic patch isthe LECTRO PATCH™ sold by General Medical Company of Los Angeles, Calif.The patch provides dosages of different concentrations which can becontinuously or periodically administered across the skin usingelectronic stimulation of reservoirs containing the inhibitors oractivators. Topical administration also includes epidermaladministration which involves the mechanical or chemical irritation ofthe outermost layer of the epidermis sufficiently to provoke an immuneresponse to the irritant. The irritant attracts APCs to the site ofirritation where they can then take up the inhibitor or activator. Oneexample of a mechanical irritant is a tyne-containing device. Such adevice contains tynes which irritate the skin and deliver the drug atthe same time. For instance, the MONO VACC manufactured by PasteurMerieux of Lyon, France. The device contains a syringe plunger at oneend and a tyne disk at the other. The tyne disk supports several narrowdiameter tynes which are capable of scratching the outermost layer ofepidermal cells. Chemical irritants include, for instance, keratinolyticagents, such as salicylic acid and can be used alone or in conjunctionwith mechanical irritants.

Injections can be e.g., intravenous, intradermal, subcutaneous,intramuscular, or interperitoneal. For example, the inhibitor can beinjected intravenously or intramuscularly for the treatment of multiplesclerosis, or can be injected directly into the joints for treatment ofarthritic disease, or can be injected directly into the lesions fortreatment of pemphigus vulgaris. The activator can be injectedinterdermally for treatment or prevention of infectious disease, forexample. In some embodiments, the injections can be given at multiplelocations. Implantation includes inserting implantable drug deliverysystems, e.g., microspheres, hydrogels, polymeric reservoirs,cholesterol matrixes, polymeric systems, e.g., matrix erosion and/ordiffusion systems and non-polymeric systems, e.g., compressed, fused, orpartially-fused pellets. Inhalation includes administering the inhibitoror activator with an aerosol in an inhaler, either alone or attached toa carrier that can be absorbed. For systemic administration, it may bepreferred that the inhibitor or activator is encapsulated in liposomes.

In certain embodiments of the invention, the administration can bedesigned so as to result in sequential exposure of the inhibitor oractivator over some period of time, e.g., hours, days, weeks, months oryears. This can be accomplished by repeated administrations of theinhibitor or activator, by one of the methods described above, oralternatively, by a controlled-release delivery system in which theinhibitor or activator is delivered to the mammal for a prolonged periodwithout repeated administrations. By controlled-release delivery system,it is meant that total release of the inhibitor or activator does notoccur immediately upon administration, but rather is delayed for someperiod of time. Release can occur in bursts or it can occur graduallyand continuously. Administration of such a system can be, e.g., bylong-lasting oral dosage forms, bolus injections, transdermal patches,and subcutaneous implants.

Examples of systems in which release occurs in bursts includes, e.g.,systems in which the inhibitor or activator is entrapped in liposomeswhich are encapsulated in a polymer matrix, the liposomes beingsensitive to specific stimuli, e.g., temperature, pH, light or adegrading enzyme and systems in which the inhibitor or activator isencapsulated by an ionically-coated microcapsule with a microcapsulecore degrading enzyme. Examples of systems in which release of theinhibitor is gradual and continuous include, e.g., erosional systems inwhich the inhibitor or activator is contained in a form within a matrixand effusional systems in which the inhibitor or activator permeates ata controlled rate, e.g., through a polymer. Such sustained releasesystems can be e.g., in the form of pellets, or capsules.

The inhibitor or activator can be suspended in a liquid, e.g., indissolved form or colloidal form. The liquid can be a solvent, partialsolvent, or non-solvent. In many cases, water or an organic liquid canbe used.

The inhibitor and activator are administered to the mammal in atherapeutically-effective amount. The therapeutically-effective amountis meant that amount which is capable of at least partially preventing,reversing, reducing, decreasing, ameliorating, or otherwise suppressingthe particular immune response being treated, when an inhibitor isadministered or inducing, activating, or otherwise increasing aparticular immune response when an activator is administered. Atherapeutically-effective amount can be determined on an individualbasis and will be based, at least in part, on consideration of thespecies of mammal, the mammal's age, sex, size, and health; theinhibitor or activator used, the type of delivery system used; the timeof administration relative to the severity of the disease; and whether asingle, multiple, or controlled-release dose regiment is employed. Atherapeutically-effective amount can be determined by one of ordinaryskill in the art employing such factors and using no more than routineexperimentation.

In some embodiments, the concentration of the inhibitor or activator ifadministered systemically is at a dose of about 1.0 mg to about 2000 mgfor an adult of 70 kg body weight, per day. In other embodiments, thedose is about 10 mg to about 1000 mg/70 kg/day. In yet otherembodiments, the dose is about 100 mg to about 500 mg/70 kg/day.Preferably, the concentration of the inhibitor or activator, if appliedtopically, is about 0.1 mg to about 500 mg/gm of ointment or other base,more preferably about 1.0 mg to about 100 mg/gm of base, and mostpreferably, about 30 mg to about 70 mg/gm of base. The specificconcentration partially depends upon the particular inhibitor oractivator used, as some are more effective than others. The dosageconcentration of the inhibitor or activator actually administered isdependent at least in part upon the particular immune response beingtreated, the final concentration of inhibitor or activator that isdesired at the site of action, the method of administration, theefficacy of the particular inhibitor or activator, the longevity of theparticular inhibitor or activator, and the timing of administrationrelative to the severity of the disease. Preferably, the dosage form issuch that it does not substantially deleteriously effect the mammal. Thedosage can be determined by one of ordinary skill in the art employingsuch factors and using no more than routine experimentation.

The lysosomal UCP inhibitors of the invention can be administered incombination with other therapeutics. For instance the inhibitors can beadministered in combination with anti-infective agents. Antibioticswhich are effective in treating a wide to variety of diseases in humansand animals including tuberculosis (caused by Mycobacteriumtuberculosis, Mycobacterium bovis, and other Mycobacteria), leprosy,Mycobacterium avium complex (MAC) infections, Mycobacterium marinuminfection, Mycobacterium fortuitum infection, Mycobacterium Kansaiiinfection, brucellosis, Q fever, tularemia, salmonellosis, typhoidfever, Yersinia infections (including Y. pestis, Y. enterocolitica andY. pseudotuberculosis), ehrlichiosis, chlamydiosis (including C.psittaci, C. trachomatis), histoplasmosis, toxoplasmosis andleishmaniasis, include but are not limited to rifampin, rifabutin,isoniazid, ethambutol, pyrazinamide, thiacetazone, para-aminosalicylicacid, aminoglycosides (including gentamycin, streptomycin, amikacin,kanamycin, viomycin, capreomycin, quinolones (including ciprofloxacin,ofloxacin), ethionamide, prothionamide, cycloserine, dapsone,clofazimine, sparfloxacin, minocycline, clarithromycin, azithromycin,doxycycline, cefoxitin, tetracyclines, cefotaxime, fluoroquinolones,ceftriaxone, chloramphenicol, trimethaprim-sulfamethoxazole, ampicillin,sulfonamides, amoxicillin, ketoconazole, itraconazole, fluconazole,pyrimethamine sulfadiazine, clindamycin, atovaquone sodiumstibogluconate, antimonials, amphotericin B, pentamidine, polymixindefinsins and other peptide antibiotics used to treat intracellularpathogens. Preferred antibiotics are those which are designed to treatdiseases that are caused by intracellular pathogens.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not to be construed aslimiting the present invention to these examples. As will be apparent toone of ordinary skill in the art, the present invention will findapplication in a variety of compositions and methods.

EXAMPLES Example 1 Metabolic State of a Cell is Indicative of CellSurface Fas Expression and Sensitivity/Resistance to Cell Death

1. Resistance to apoptosis is characterized by failure to express Fas:The cell lines utilized herein include L1210, a leukemic cell line;HL60, a human pro-myelocytic cell line; and PC12, a pheochromocytomacell line which can be induced to differentiate into a neuronal cellline in the presence of NGF (Lindenboim, L, et al., Cancer Res, 1995,55:1242-7). Each cell line was examined in parallel with apoptoticresistant sublines: L1210 DDP, HL60 MDR, and PC12Trk. L1210 DDP areresistant to cisplatin and methotrexate; HL60 MDR are resistant toadriamycin induced apoptosis; PC12 to TrkA, which have been transfectedwith TrkA which results in constitutively expression the NGF receptors,are not susceptible to alcohol and NGF withdrawal as are the PC12 cells.

The apoptosis sensitive cells from each tissue origin weremorphologically round, non-adherent, rapidly dividing cells, with theexception of the PC12 cell line. The apoptosis resistant cells from alltissue origins were morphologically large, adherent, and slowly dividingcells.

The recently characterized molecules, Fas (CD95) and Fas Ligand (CD95L),have been strongly implicated in the process of apoptotic death (Muller,M, et al., J Clin Invest, 1997, 99:403-413). We examined expression ofFas on the above-identified cell lines. Flow cytometric analysis of Fasexpression was performed using an isotype versusFITC-anti-Fas(Pharmingen) on L1210; PC12; and HL60 cells and resistantcell lines L1210DDP, PC12Trk; and HL60MDR. A Coulter Epics Elite flowcytometer with a single excitation wavelength (488 nm) and band filtersfor PE (575 nm), FITC (525 nm) and Red613 (613 nm) was used to analyzethe stained cells. Each sample population was classified for cell size(forward scatter) and complexity (side scatter), gated on a populationof interest and evaluated using 40,000 cells. Criteria for positivestaining were established by comparison with the intensity of theisotype controls, thick lines. Independent of tissue origin, all of theapoptosis resistant lines fail to express cell surface Fas bothconstitutively and in the presence of agents that induce apoptosis inthe parental cell lines.

2. Resistance to apoptosis is characterized by relatively high rates ofglucose oxidation and utilization: We performed experiments to examinethe correlation between cell surface Fas expression and glucosemetabolism. As a prototype for the Fas positive and Fas negative cellswe used the L1210 and the L1210DDP cell lines, as Fas positive and Fasnegative, respectively. We directly measured the rates of glucoseutilization and oxidation of L1210 and L1210DDP. Ratios were generatedby using nanomolar values.

Rate of glucose utilization was measured by the method of Ashcroft etal. Briefly, cells were incubated 90 mM at 37° C. in 100 μl KRB, glucose(5.5 mM), 1.3 μCi to D-[5-³H] glucose (Amersham, Arlington Heights,Ill.). The reaction was carried out in a 1 ml cup contained in a rubberstoppered 20 ml scintillation vial that had 500 μl of distilled watersurrounding the cup. Glucose metabolism was stopped by injecting 100 μl1 M Hcl through the stopper into the cup. An overnight incubation at 37°C. was carried out to allow equilibration of the [³H]-H₂O in thereaction cup and the distilled water, followed by liquid scintillationcounting of the distilled water.

Rate of glucose oxidation was measured by incubating cells for 90 min at37° C. in 100 ml of reaction buffer, glucose (2.8, 8.3, 27.7 mmol/l),1.7 mCi (U-14C glucose). The reaction was carried out in a 1 ml cup in a20 ml scintillation vial capped by a rubber stopper with a center wellthat contains filter paper. Metabolism was stopped and CO₂ liberatedwith 300 ml 1 mol/l HCl injected through the stopper into the cupcontaining the cells. CO₂ was trapped in the filter paper by injecting10 ml 1 mol/l KOH into the center well, followed 2 hours later by liquidscintillation counting. Tubes containing NaHCO₃ and no cells were usedto estimate the recovery of ¹⁴CO₂ in the filter paper, routinely closeto 100%.

The results are presented in Table 1.

TABLE 1 Glucose Metabolism in L1210/0 and L1210/DDP L1210/0 L1210/DDPGlucose Utilization 1740 ± 920 3470 ± 460  (pmol glucose/90 min/ 50,000cells) Glucose Oxidation 235 ± 7  428 ± 124 (pmol glucose/90 min/ 50,000cells) Glucose Utilization/Oxidation 7.4 8.1

Because the L1210 and L1210DDP cells are tumor cell lines and are likelyto have increased ratios of glucose oxidation to utilization (Warburg,O, et al., Klin Woch, 1926, 5:829-832), we measured glucose utilizationon normal lymphocytes. We isolated 10⁶ splenic lymphocytes from C57BL/6animals, Fas-deficient C57BL/6 (B6.lpr), and FasL defective C57BL/6(B6.gld) animals. The rate of glucose utilization and oxidation of theFas deficient and the FasL deficient lymphocytes are demonstrated inTable 2. The ratio of glucose utilization to oxidation is highest in lprlymphocytes and lowest in wild type normal, quiescent lymphocytes.

TABLE 2 Glucose Metabolism in Lymphocytes from Normal, Fas Deficient andFasL Deficient Mice GLUCOSE UTILIZATION/ CELL TYPE RATIO GLUCOSEOXIDATION b6 lpr gld GLUCOSE UTILIZATION 0.04 0.36 0.22 (nmol glucose/90mins/50,000 cells) GLUCOSE OXIDATION 73.24 164.51 122.82 (pmolglucose/90 mins/50,000 cells)

These data (Table 1 & 2) demonstrate high rates of glucose utilizationand oxidation of both tumor lines relative to the normal lymphocytes;and higher rates of glucose utilization and oxidation of the apoptoticresistant line relative to the wild type. There is an importantdifference in the ratio of glucose utilization to oxidation betweennormal and Fas or FasL deficient animals, with the ratio being higherfor lymphocytes from both mutant strains of animals. The consequences ofuncoupling are a decrease in mitochondrial membrane potential; use offat as a carbon source increased rate of glycolysis, increased rate ofelectron transport, and energy dissipation, in a form other than ATP.These data suggest that there is an increase in proton leak in the cellswith high rates of glucose oxidation and utilization relative to thenormal cells, suggesting some degree of uncoupling may have occurred inthese cells.

3. Fas Expression Increases as a Function of Glucose: We investigatedthe effect of increasing concentrations of glucose on cell surface Fasexpression. L1210 and L1210/DDP cells were cultured in glucose free RPMImedia or in media supplemented with insulin and glucose for 16 hours.Intra- and extracellular Fas expression was to determined by labelingthe cells with FITC-conjugated anti-Fas antibodies (Pharmingen), orFITC-conjugated isotype control, then subtracting the fluorescenceintensity of the isotype staining from Fas staining for each treatmentgroup.

These data showed that Fas expression increases as a function of glucoseconcentration and that as a result the cell surface Fas negativeL1210/DDP begin to express cell surface Fas.

4. Treatment of L1210 DDP cells with staurosporin restores Fasexpression and susceptibility to drug-induced apoptosis: L1210, but notL1210 DDP, undergo apoptotic cell death. We treated L1210 or L1210 DDPcells with the staurosporin, which inhibits protein kinase C andincreases mitochondrial membrane potential, or an anti-cancer agent towhich both cells are sensitive, adriamycin. Fas expression was increasedor induced on both L1210 and L1210 DDP, respectively, in the presence ofstaurosporin or adriamycin. The L1210 DDP changed morphologically andbegan to divide rapidly, changes which appeared to correspond with areversion back to the phenotype of the L1210 cells. These resultsdemonstrate that Fas expression results in parallel with alteredmetabolic activity.

5. Confocal microscopy reveals that resistance to apoptosis ischaracterized by intra-(but not extra) cellular Fas expression: L1210DDP cells express no cell surface Fas. To address the possibility thatFas is expressed, but has been targeted to a subcellular organelle, wepermeabilized and stained L1210 and L1210DDP cells with fluorochromeconjugated anti-Fas antibody (J02.2, Pharmingen). The cells wereexamined by confocal microscopy. (This experiment was representative offour experiments).

Our data indicate that L1210 DDP cells express Fas in an intracellular,cytosolic compartment. Fluorochrome-conjugated isotype matched antibodywas used as control. Additionally, these data also demonstrate that theFas negative, apoptosis resistant cells, express intracellular Fas.

6. Fas-deficient (lpr) lymphocytes express intra- (but not extra-)cellular Fas molecules: We isolated lymphocytes from spleens of C57BL/6mice and from C57BL6 transgenics having the lpr mutation (loss of Fassensitivity). Cells were stained with to fluorescein conjugated hamsteranti-Fas and examined by confocal microscopy.

Results demonstrate that unstimulated, non-permeabilized splenocytesfrom C57BL/6 animals express Fas at low levels relative to isotypecontrols. Interestingly, significant levels of Fas expression weredetected in permeabilized normal lymphocytes. As expected,non-permeabilized cells from C57BL6.lpr animals express no detectablecell surface Fas relative to isotype control. Interestingly,intracellular Fas staining of permeabilized splenocytes from C57B1/6.lpranimals reveals intracellular expression of Fas. These resultsdemonstrate that mutations affecting susceptibility to Fas-induced deathprevent cell surface, but not intracellular expression of the Fasmolecule.

7. Anti-cancer agents induce susceptibility to Fas-induced cell death:To determine if the anti-cancer agent methotrexate sensitizes L1210 orL1210/DDP cells to Fas induced cell death, we cultured L1210 cells inthe presence or absence of 10⁻⁸ M methotrexate for 72 hours. Each groupof cells was cultured on uncoated plates or plates coated with 10 g/mlanti-Fas (Jo.2.2, Pharmingen). We analyzed cell death using flowcytometry. Forward angle and 90 degree light scatter were used todistinguish between live and dead cells. Dead cells were gated asforward angle light scatter low/high ethidium bromide retaining cells.Percent death was calculated over the total number of cells acquired. InTable 3 below, values indicate % dead cells over background of untreatedcells.

TABLE 3 Fas-induced cell death L1210/0 L1210/13DP Control 4.72 40.88anti-Fas 79.98 46.60 Coated Plates

Additionally, L1210 and L1210/DDP cells were treated with 10-8 Mmethotrexate for 24 hours. Flow cytometric analysis revealed twopopulations based on forward side scatter. The forward scatter highpopulations did not take ethidium bromide and were therefore viable. Theforward scatter low populations took up ethidium bromide differentially.The L1210 cells took up a moderate amount. Analysis of DNA fragmentsreveals that L1210 produced a ladder of nucleosome sized fragmentsindicative of apoptosis, whereas L1210/DDP cells did not. This latterphenotype—loss in forward scatter and membrane permeability with no “DNAladdering”—is the hall mark of oncosis.

8. Fas Deficient Lymphocytes are also drug resistant to methotrexate: Weisolated splenic lymphocytes from aged-matched wild type C57BL/6 miceand C57BL6.lpr and C57BL.gld. Splenocytes from C57BL/6 lpr or gldanimals were isolated, red cells depleted, and single cell suspensionsprepared. Cells were cultured in the absence or presence of 5×10⁻⁸ Mmethotrexate for 18 or 32 hours. Cells were harvested and viability wasdetermined by flow cytometric analysis and confirmed with trypan blueexclusion.

The data demonstrate decreased susceptibility to methotrexate-inducedapoptosis in Fas deficient lymphocytes. These data are consistent withthe notion that Fas is required for drug susceptibility.

9. Drug resistant cells express intracellular fas, UCP and bcl-2: Wedetermined if wild type and/or drug resistant cells expressintracellular and surface fas, UCP and bcl-2. We stainednon-permeabilized L1210 and L1210/DDP cells for cell surface orintracellular Fas. The data show that while there is no cell surfaceexpression of Fas on the drug/apoptotic resistant cells, the drugresistant cells express high levels of intracellular Fas. The drugresistant cells are cell surface Fas negative and protected from deathresulting from changes in mitochondrial membrane permeabilitytransitions.

10. Cell surface and Intracellular Fas Levels in Melanoma Cells.

B16 cells were cultured in the in the presence of differentconcentrations of sodium acetate and Fas expression was measured. Thedata showed the level of cell surface Fas expression onnon-permeabilized and intracellular Fas expression in permeabilized B16melanoma cells. With increasing concentrations of sodium acetate, thelevels of intracellular Fas declined and the levels of cell surface Fasincreased, demonstrating a translocation of Fas from intracellularstores to the surface.

The rates of glucose utilization and oxidation in B16 melanoma cellsalso was determined. Again cells were cultured in the presence ofvarying concentration of sodium acetate. Both glucose utilization andglucose oxidation (measured in nmoles) decreased with increasingconcentrations of sodium acetate, demonstrating a correlation withexpression of cell surface Fas in the same cells.

11. Sodium Acetate Increases Cell Surface Fas Expression.

Sodium Acetate as a mitochondrial modifying agent was examined. L1210 orL1210DDP cells were cultured in the presence of graded concentrations ofsodium acetate in the medium. Cells were stained with Jo2.2, afluorescein conjugated anti-Fas antibody, or an isotype control. Cellsurface staining was measured flow cytometrically. The percentage ofmean fluorescence intensity over the isotype control was plotted. Thedata indicate that the presence of acetate increases cell surface Fasexpression in both cell lines.

The effects of acetate on susceptibility to Fas-dependent cell deathwere also examined. Cells cultured with acetate were loaded with 51 Crand plated onto FasLbearing or mock transfected fibroblast to determinesensitivity to Fas-induced cell death. The data indicate that in a dosedependent manner, culture of both cell types with acetate resulted insusceptibility to Fas-dependent cell death.

Example 2 Metabolic State of a Cell is Indicative of Cell Surface andIntracellular UCP Expression and Sensitivity/Resistance to Cell Death

1. Intracellular UCP is Present in a Panel of Tumor Cells.

We extended our analysis of intracellular expression of UCP to othertumor cells. Intracellular UCP expression was examined flowcytometrically on cells which had been permeabilized and stained asindicated. The histograms represent FITC isotype control versus stainedwith Rabbit anti-UCP (a kind gift of Mary Ellen Harper)FITC-anti-Rabbit. A Coulter Epics Elite flow cytometer with a singleexcitation wavelength (488 nm) and band filters for PE (575 nm), FITC(525 nm) and Red613 (613 nm) was used to analyze the stained cells. Eachsample population was classified for cell size (forward scatter) andcomplexity (side scatter), gated on a population of interest andevaluated using 40,000 cells. Criteria for positive staining wereestablished by comparison with isotype controls, thin lines to specificstain, thick lines.

All of the tumor cells lines examined express UCP intracellularly. Thesedata are consistent with the idea that expression of UCP in tumor cellsis generalizable to all tumor cells, and likely results from the welldocumented shift in subcellular production of ATP from mitochondria tocytosol as cells divide. Importantly, these data also demonstrate thatexpression of UCP2 is not specific to lymphoid tumors. The L929 cellsare fibroblasts and the PC 12 Trk cells which are derived frompheochromocytoma cell lines, respectively. The EL4 cells are a mousethymoma cell line and Jurkat are human T cell tumor cells.

To confirm that flow cytometrically detected UCP expression wasmitochondrial, we isolated mitochondria from L1210 and L1210 DDP, andperformed Western Blot analysis blotting with rabbit anti-UCPantibodies. Mitochondria were isolated using differential centrifugationas adapted from (Reinhart, P H, Taylor, W M and Bygrave F L (1982) Aprocedure for the rapid preparation of mitochondria from rat liver.Biochem. J. 204: 731-735. and Sims N R (1990) Rapid isolation ofmetabolically active mitochondria from rat brain and subregions usingPercoll density gradient centrifugation. J. Neurochem. 55:698-707.) Thefollowing samples were run: molecular weight markers (BIORADBiotinylated SDS-PAGE standards; L1210/0 mitochondrial protein (40:g)from two distinct mitochondrial preparations; L1210/DDP mitochondrialprotein (40:g) from two distinct mitochondrial preparations; and UCPstandard (0.75:g) from rat brown adipose tissue (which expresses UCPs1-3). Rabbit anti-hamster UCP was used at a dilution of 16,000. Thesecondary antibody: goat anti-rabbit IgG conjugated to HRP at 1:10,000.Chemiluminescent detection: Amersham ECL kit.

The blot showed greater levels of mitochondrial UCP in the drugresistant L1210/DDP than in L1210/0. The detected mitochondria proteinhas an approximate molecular weight of 30 kDa, close to the predictedmolecular weight of UCP2 (33 kDa).

To determine whether increased UCP corresponded to increasedmitochondrial proton leak and a lower mitochondrial membrane potential(Δψm) we assessed characteristics of non-phosphorylating respiration inintact L1210 wild type and L1210 DDP cells. State 4 Δψm in DDP cells, xmV, was significantly lower than in wild type cells, y mV (p<0.001), andstate 4 oxygen consumption in DDP cells is significantly higher than inwild type cells, indicating increased mitochondrial proton leak.

2. Cell Surface UCP is Present in a Panel of Tumor Cells.

The cell lines utilized herein include L1210, a leukemic cell line; andHL60, a human pro-myelocytic cell line. Each cell line was examined inparallel with apoptotic resistant sublines: L1210 DDP, and HL60 MDR.L1210 DDP are resistant to cisplatin and methotrexate and HL60 MDR areresistant to adriamycin induced apoptosis. The apoptosis sensitive cellsfrom each tissue origin were morphologically round, non-adherent,rapidly dividing cells. The apoptosis resistant cells from all tissueorigins were morphologically large, adherent, and slowly dividing cells.

We examined expression of UCP on the plasma membrane of theabove-identified cell lines to determine if wild type and/or drugresistant cells express intracellular and surface UCP. We stainednon-permeabilized L1210 and L1210/DDP cells for cell surface orintracellular UCP. Flow cytometric analysis of UCP expression wasperformed using an isotype versus FITC-anti-UCP (described above) onL1210 and HL60 cells and resistant cell lines L1210DDP and HL60MDR. ACoulter Epics Elite flow cytometer with a single excitation wavelength(488 nm) and band filters for PE (575 nm), FITC (525 nm) and Red613 (613nm) was used to analyze the stained cells. Each sample population wasclassified for cell size (forward scatter) and complexity (sidescatter), gated on a population of interest and evaluated using 40,000cells. Criteria for positive staining were established by comparisonwith the intensity of the isotype controls, thick lines.

The apoptosis resistant lines failed to express cell surface UCP(HL60MDR) or expressed lower levels of UCP than the wild type cells(L1210DDP). Each of the wild type cell lines expressed high levels ofcell surface UCP. The data demonstrate a correlation between fas and UCPexpression. The data described above demonstrate that while there is nocell surface expression of Fas on the drug/apoptotic resistant cells,the drug resistant cells express high levels of intracellular Fas,similar to UCP. The drug resistant cells are cell surface Fas negativeand protected from death resulting from changes in mitochondrialmembrane permeability transitions.

3. Cell Surface and Intracellular UCP and Fas Levels in Melanoma Cells.

B16 cells were cultured and either permeabilized or unpermeabilized andUCP expression was measured. The data showed the absence of cell surfaceUCP expression on non-permeabilized B16 cells, similar to drug resistanttumor cells described above. Intracellular UCP expression inpermeabilized B16 melanoma cells, on the other hand, was very high.

B16 cells also were cultured in the presence of different concentrationsof sodium acetate and Fas expression was measured. The data showed thelevel of cell surface Fas expression on non-permeabilized andintracellular Fas expression in permeabilized B16 melanoma cells. Withincreasing concentrations of sodium acetate, the levels of intracellularFas declined and the levels of cell surface Fas increased, demonstratinga translocation of Fas from intracellular stores to the surface.

The rates of glucose utilization and oxidation in B16 melanoma cellsalso was determined. Again cells were cultured in the presence ofvarying concentration of sodium acetate. Both glucose utilization andglucose oxidation (measured in nmoles) decreased with increasingconcentrations of sodium acetate, demonstrating a correlation withexpression of cell surface Fas in the same cells.

4. UCP-2 mRNA Levels in Tumor Cells.

mRNA was isolated from L1210 wild type cells, L1210-DDP cells, apositive control (brown adipose fat tissue from a UCP-1 negative (−/−)mouse), and a negative control (brown adipose fat tissue from a UCP-1positive/negative (+/−) mouse) and processed by Northern blot analysis.L1210 wild type cells expressed the highest amounts of UCP-2 mRNA, evenhigher than positive control cells. L1210-DDP cells expressed much lowerlevels of UCP-2 than wild type or the positive control cells.

5. UCP Protein Levels in Tumor Cells.

Protein was isolated from plasma membrane of L1210 wild type cells andL1210-DDP cells and processed by Western blot analysis. L1210 wild typecells expressed the highest amounts of plasma membrane UCP. L1210-DDPcells expressed much lower levels of plasma membrane UCP than wild type.

Example 3 Exposure of Chemotherapy Sensitive Tumor Cells but notChemotherapy Resistant Cells to Anti-UCP Antibody Causes Increased CellDeath

Chemotherapy-sensitive cells HL60 and chemotherapy resistant cellsHL60-MDR were exposed to a labeled anti-UCP antibody (described above)for two 15 minute intervals and subjected to flow cytometry, asdescribed above.

Scatter plots were generated for each sample. In the first plot theforward versus side scatter of untreated HL60 cells represents apopulation of healthy living cells. When HL60s were treated with theanti-UCP antibody, the forward scatter decreased and the side scatterincreased demonstrating a higher number of dead cells present in thepopulation. It is expected that longer incubation times with theanti-UCP antibody will result in a greater number of dead cells withinthe population. There was no difference between the anti-UCP untreatedand treated HL60-MDR cells. The lack of effect of the UCP antibody onHL60MDR cells was expected, since these cells do not express cellsurface UCP.

Example 4 Pancreatic B Cells Express Mitochondrial UCP and have No CellSurface Fas

1. Loss of antigen in β-cell tumors: Proliferation with two responder Tcell clones, BDC-2.5 and BDC-6.9, was tested using NOD peritoneal cellsas APC and as antigen, either freshly prepared NOD islet cells (control)or β tumor cells, or NIT-1, an established beta tumor cell line from theNOD-RIPTag mouse. Upon harvesting the islet tumors, the β-cells obtainedare fully as antigenic as normal NOD islet cells. The NIT-1 line is alsoantigenic for these T cell clones, but only at low passage numbers; withcontinued culture, the line changes its morphology and growth kineticsand undergoes complete loss of antigen.

2. Response of pancreatic β-cells to glucose: The experiments describedbelow were designed to test the hypothesis that β cell metabolism may belinked to immune recognition and destruction. Glucose utilization wasmeasured as [³H]H₂O production from 5-[³H] glucose in normal rat islets.Glucose oxidation was measured as [¹⁴C]CO₂ production from U-[¹⁴C]glucose. The data show increasing glucose utilization and oxidation inβ-cells as a function of increasing glucose concentration.

3. Normal β-cells Express Intracellular UCP2 and No Cell Surface Fas:Normal β-cells have a specialized glucose response which is based on thecell being responsive to physiologic glucose concentrations. The processthat mediates the glucose responsiveness is the process involving fluxthrough glycolysis. β-cell glucose usage is mediated through arelatively unique system that entails specialized high K_(m) glucosetransporter (GLUT2) and glucose phosphorylation isoforms (glucokinase).We isolated β-cells from C3H mice, stained the isolated cells withanti-Fas, and electronically gated viable cells. In parallel, cells werepermeabilized and stained with an antibody to UCP2 (kindly provided byDrs. Jean Himms-Hagen and M. E. Harper). The data indicated that normalβ-cells expressed intracellular UCP2 and no cell surface Fas.

4. Fas Expression and Mitochondrial Membrane Potential are a Function ofGlucose Concentration in Mouse β Cells.

The central question is whether Fas expression is altered by changes inphysiological glucose concentrations in normal β cells and does themitochondrial membrane potential increase, suggesting that the cell hasATP synthesis resulting from increased rates of electron transport.Islets were isolated and dispersed with trypsin and a cell strainer.Debris and dead cells were removed and applying the cells to a 1.066Percoll gradient. Electronic gating of the cells was used to segregatethe populations of islets cells. The region with larger cells were gatedβ cells where the region with smaller cells were gated as alpha cells.Other larger cells were excluded because they contained δ cells. Thecells were treated overnight with either physiological 11.1 mM glucoseor high glucose 55.5 mM glucose. Fas expression was determined bystaining with a FITC conjugated antibody. Mean fluorescence of stainingwith isotype control antibody was subtracted. Measurement ofmitochondrial membrane potential was measured using JC-1 as afluorescence probe. The relative membrane potential was read by takingthe red mean fluorescence (aggregated JC-1 labeled) divided by meangreen fluorescent (monomeric JC-1) labeled fluorescence. Our datasuggest that as glucose concentration increases, the large β cell subsetof gated cells have increased Fas expression and concomitant increasedmitochondrial membrane potential, while the smaller (possibly alpha,glucagon producing cells) do not.

5. Determination of Mitochondrial Membrane Potential in β Cells Isolatedfrom Four Strains of Animals.

Mitochondrial membrane potential is assessed flow cytometrically usingmitotracker red. The amount of membrane potential was measured in thefour strains of animals AB-, AB-Ea, C57B1/6, BITgEa, described in moredetail below. Mitochondrial membrane potential was highest in theAB-strain, followed by the AB-EA stain. The C57B1/6 and BITgEa strainshad much lower mitochondrial membrane potential.

Example 5 Overexpression of UCP2 Shifts UCP from Plasma Membrane andGrowth Arrests Cells

Overexpression of UCP in L1210 and U937 cells induced a shift tointracellular UCP and caused a phenotypic change to the cells. Thesecells became growth arrested and chemotherapy resistant. UCP2 alsoprevented free radical production, thus preventing respiratory burst.Cells transfected with a mock vector remained cell surface UCP positive,chemotherapy sensitive and demonstrated no change with respect toreactive oxygen production.

Example 6 UCP is Expressed in Lysosomal Membranes

UCP stained cells were subjected to confocal microscopy and examined forUCP localization. Surprisingly, UCP was observed in lysosomes of rapidlydividing, drug sensitive cells.

Example 7 Inhibition of Lysosomal Translocation

Tunicamycin was used to demonstrate an inhibition of lysosomal UCPtranslocation. MDR cells, which are plasma membrane UCP negative andlysosomal UCP positive were treated with tunicamycin. Treatment with thedrug caused a shift in the intracellular UCP from the lysosome andmitochondria to the plasma membrane. These same cells were treated withtunicamycin and an-anti-UCP2 antibody. This combination therapy inducedcell death, as a result of the expression of UCP on the plasma membrane.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

1. A method for inhibiting plasma membrane UCP expression in a cell, comprising: contacting a cell with a plasma membrane UCP inhibitor to inhibit plasma membrane UCP expression, wherein the plasma membrane UCP inhibitor is selected from the group consisting of a UCP binding peptide or molecule, an anti-UCP antibody, a hydrophobic nucleotide analog, and a non-omega-3 fatty acid. 2.-37. (canceled)
 38. A method for sensitizing a resistant tumor cell to a cytotoxic therapy, comprising: expressing a functional UCP or UCP fragment in a plasma membrane of a resistant tumor cell to sensitize the resistant tumor cell to a cytotoxic therapy.
 39. The method of claim 38, wherein the method is performed in a subject having a cancer. 40.-55. (canceled)
 56. A method for inducing cellular division in a growth arrested cell, comprising: expressing a functional UCP or UCP fragment in a plasma membrane of a growth arrested cell to induce cell division of the cell.
 57. The method of claim 56, wherein the growth arrested cell is a nerve cell.
 58. The method of claim 57, wherein the functional UCP or UCP fragment is administered to a subject in conjunction with a delivery vehicle.
 59. The method of claim 58, wherein the delivery vehicle is a liposome.
 60. A method for regulating lysosomal pH, comprising: modifying lysosomal UCP activity in a cell to regulate lysosomal pH. 61.-62. (canceled)
 63. The method of claim 60, wherein the lysosomal UCP activity is modified by contacting the cell with a lysosomal UCP inhibitor.
 64. The method of claim 63, wherein the lysosomal UCP inhibitor is selected from the group consisting of a dominant negative lysosomal UCP, and a lysosomal targeted binding peptide or molecule.
 65. The method of claim 60, wherein the lysosomal UCP activity is modified by contacting the cell with a lysosomal UCP activator.
 66. A method for treating or preventing an infectious disease, comprising: administering to a subject having or at risk of developing an infectious disease a lysosomal UCP inhibitor in an effective amount for treating or preventing the infectious disease.
 67. The method of claim 66, wherein the lysosomal UCP inhibitor is selected from the group consisting of a dominant negative lysosomal UCP, and a lysosomal targeted binding peptide or molecule.
 68. The method of claim 66, further comprising administering an antigen to the subject.
 69. The method of claim 68, wherein the antigen is selected from the group consisting of a viral, a bacterial, a parasitic, and a fungal antigen.
 70. The method of claim 66, wherein the subject is infected with an intracellular pathogen.
 71. The method of claim 70, wherein the intracellular pathogen is an intracellular bacteria.
 72. The method of claim 70, wherein the intracellular pathogen is an intracellular parasite.
 73. A method for treating autoimmune disease, comprising: administering to a subject having autoimmune disease a UCP activator in an effective amount to prevent antigen presentation.
 74. The method of claim 73, wherein the UCP activator is a functional UCP or UCP fragment with a lysosomal membrane targeting molecule. 